System and method for automated single cell processing

ABSTRACT

A system and method for automated single cell capture and processing is described, where the system includes a deck supporting and positioning a set of sample processing elements; a gantry for actuating tools for interactions with the set of sample processing elements supported by the deck; and a base supporting various processing subsystems and a control subsystems in communication with the processing subsystems. The system can automatically execute workflows associated with single cell processing, including mRNA capture, cDNA synthesis, protein-associated assays, and library preparation, for next generation sequencing.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application No. 16/867,256,filed 5 May 2020, which claims the benefit of U.S. ProvisionalApplication No. 62/844,470, filed on 7 May 2019 and U.S. Application62/866,726, filed on 26 Jun. 2019, which are each incorporated in itsentirety herein by this reference.

TECHNICAL FIELD

This invention relates generally to the cell capture and cell processingfield, and more specifically to a new and useful automated system andmethod for single cell capture and processing in the cell capture andcell processing field.

BACKGROUND

With an increased interest in cell-specific drug testing, diagnosis, andother assays, systems and methods that allow for individual cellisolation, identification, and retrieval are becoming highly desirable.Single cell capture systems and methods have been shown to beparticularly advantageous for these applications. However, associatedprocesses and protocols for single cell capture and subsequent analysisoften must be performed in a particular order and with a high precisionin order to properly maintain the cells. As such, these processes can betime consuming for the user, as well as result in damage to the cells orotherwise unfavorable results if they are not performed properly (e.g.,through mistakes in pipetting, through a mix-up of reagents, etc.). Inparticular, these novel high throughput single cell cytometry assayshave great utility in translational medicine, personalized therapyselections, clinical diagnostics, and/or other applications of use, butlack of automation prevents proper performance by novice users, therebylimiting throughput.

Thus, there is a need in the cell capture and cell processing field tocreate a new and useful system and method for single cell capture andprocessing.

BRIEF DESCRIPTION OF THE FIGS.

FIGS. 1A-1D depict schematic representations of an embodiment of asystem for automated single cell sample processing;

FIGS. 2A-2F depict views of a variation of the system shown in FIGS.1A-1D;

FIGS. 3A-3C depict variations of a reagent cartridge associated with asystem for automated single cell sample processing;

FIGS. 4A-4C depict views of a variation of a sample processing cartridgeassociated with a system for automated single cell sample processing;

FIGS. 5A-5C depict operation modes of a lid-opening tool associated withthe sample processing cartridge shown in FIGS. 4A-4C;

FIGS. 6A-6B depict operation modes of a valve and heating subsystemassociated with the sample processing cartridge shown in FIGS. 4A-4C;

FIGS. 7A and 7B depict variations of a tool container and contentsassociated with a system for automated single cell sample processing;

FIGS. 8A-8I depict variations of features for retaining elements at adeck of a system for automated single cell sample processing;

FIG. 9 depicts an example of a fluid level detection subsystem of asystem for automated single cell sample processing;

FIGS. 10A-10C depict variations of a first subset of components used formaterial separation in a system for automated single cell sampleprocessing;

FIGS. 11A-11B depict variations of a second subset of components usedfor material separation in a system for automated single cell sampleprocessing;

FIGS. 12A-12J depict operation modes of a separation subsystemassociated with a system for automated single cell sample processing;

FIGS. 13A-13D depict views of components of a variation of a separationsubsystem associated with a system for automated single cell sampleprocessing;

FIGS. 14A-14C depict embodiments of functional coupling betweencomponents of a system for automated single cell sample processing;

FIG. 15 depicts a flow chart of an embodiment of a method for automatedsingle cell sample processing;

FIGS. 16 depicts a first variation of a method for automated single cellsample processing;

FIGS. 17 depicts a second variation of a method for automated singlecell sample processing;

FIGS. 18 depicts a third variation of a method for automated single cellsample processing; and

FIGS. 19 depicts a third variation of a method for automated single cellsample processing.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following description of the preferred embodiments of the inventionis not intended to limit the invention to these preferred embodiments,but rather to enable any person skilled in the art to make and use thisinvention.

1. Benefits

The invention(s) can confer several benefits over conventional systemsand methods.

In particular, the invention(s) confer(s) the benefit of enabling atleast partial automation of the protocols involved in single cellcapture and subsequent processing, thereby optimizing run success andconsistency. In more detail, the user can be removed from part or all ofthe method (e.g. loading samples, capping lids, on-instrument lysis,reverse transcription processes, cDNA amplification, bead or cDNAproduct retrieval, on-instrument library preparation and cleanup, etc.).Further, the system and/or method can enable better accuracy of aprotocol over conventional systems and methods (e.g. better accuracy inthe addition of the correct reagents, better temperature control ofreagents, rapid processing of critical liquid handling steps, preciseincubation times, optimal bead washing and separation, automated barcode reading, etc.). Further, the system and/or method can confer thebenefit of preventing accidents (e.g. knocking the system, spills ofreagents, contamination of sample or instrument, etc.), which cancommonly occur during the manual performance of a protocol.

Additionally, through use of limited-use and/or pre-loaded and unitizedreagent cartridges, the system and/or method can confer the benefit ofproviding a streamlined user experience with optimized quality controland design architecture to accommodate on-going development of assaysand future applications. As such, the system confers the benefit ofindependent or nearly independent control of reagents or reagent groups.In a specific example of this variation, the system includes a reagentcartridge having any or all of the following dedicated regions: a roomtemperature region, a cooling region, a heating region, a magneticregion (e.g., overlapping with a heating region), waste capture region,intermediate reagent parking region or any other suitable region. In arelated benefit, the system and/or method can confer the benefit ofenabling the user to purchase smaller volumes of reagents, such asthrough the distribution of reagents in protocol-specific types andquantities to be used in accordance with specific automated protocols.This can function to save costs, reduce reagent waste, or have any othersuitable outcome.

Additionally, through use of fluid handling and separation elements(e.g., magnetic separation components), the system and/or method canconfer the benefit of providing automated sample and library cleanupsteps. Relatedly, the system and/or method can confer the benefit ofestablishing better fluid flow throughout the system. In a firstexample, this is enabled through an automated pipetting system (e.g.,pipettor, gantry, and assorted pipette tips), which can monitor and/ordirect fluid flow (e.g., to maintain an optimal flow rate, to establishan optimal volume of reagents, etc.) without user intervention. Thefluid handling system components for single cell preparation and/orother assays may involve use of both of (a) liquid pipettor coupled to agantry for fluidic dispensing and pumping into a fluidic channel orfluidic reservoir (e.g., of a sample processing cartridge) and/or (b) abuilt-in on-chip pressurizable waste chamber connected and controlledthrough a valve integrated with the fluidic network, as described inmore detail below. Such a combined dual liquid handling system givesunprecedented control of the flow (e.g., microliter per second to tensof milliliters per second), delivery (e.g., 1-100,000 microliters), andresidence time (e.g., milliseconds to hours) of reagents through thefluidic system. Additionally or alternatively, the system can monitorand/or direct fluid flow with user intervention (e.g., with minimal userintervention, to encourage optimal user intervention, etc.).

Additionally, through software and workflow improvements, the systemand/or method can minimize number of manual operations performed by auser, and provide relevant system status reports to ensure smoothoperation and sample processing.

Additionally, in relation to sample processing disposables, the systemand/or method can confer the benefit of consolidating multiplecomponents in a manner that is scalable for disposables having a highernumber of sample processing chambers. Additionally, the system canconfer the benefit of consolidating two or more conventionally separateprocessing platform components into a single unit, which can reduce anoverall size of the system (e.g., enable a benchtop model), reduce anoverall footprint of a mechanism of the system (e.g., pipettor gantry),enable a more efficient transfer of materials among the system, orperform any other suitable function. In a specific example of thisvariation, an inlet, set of microwells, outlet valve, lid mechanism(e.g., lid of the lid mechanism, full lid mechanism, etc.) and wastechamber are all localized to a single piece.

Additionally, the invention(s) address needs in low parameter flowapplications, high parameter flow applications, mass cytometryapplications, proteogenomic applications, single cell RNA applications,protein detection applications, single cell multi-omic applications andother applications, by allowing standard users with various skill levels(e.g., novices, experts) to operate platform components. Specificworkflows implemented by embodiments of the system are described in moredetail below.

In relation to performance, the system and/or method can process cellsto generate purified libraries within a day, perform next generationsequencing (NGS) preparation, and perform other processes in astreamlined process (e.g., by a set of dedicated consumables includingan efficiently loaded reagent cartridge, a sample processing cartridge,and a container of fluid handling disposables).

Additionally, the system confers the benefit of three-dimensionalmobility of a component, such as a pipettor. In a specific example ofthis variation, the system includes a gantry providing X-Y-Z mobilityfor a pipettor, enabling the pipettor to perform a variety of tasks(e.g., piercing foil coverings of reagent tubes, transferring materialsamong a set of wells, etc.) in an automated fashion.

Additionally or alternatively, the system and/or method can confer anyother suitable benefit.

2. System

As shown in FIGS. 1A-1D, an embodiment of a system 100 for automatedsingle cell capture and processing includes: a deck no supporting andpositioning a set of sample processing elements; a gantry 170 foractuating tools for interactions with the set of sample processingelements supported by the deck no; and a base 180 supporting variousprocessing subsystems and a control subsystems in communication with theprocessing subsystems, wherein the control subsystems control states ofthe deck 110, the set of sample processing elements, and the gantry 170in order to transition the system 100 between various operation modes.Embodiments, variations, and examples of operation modes, which providevarious workflows, are described in further detail in Section 3 below.

Embodiments of the system 100 function to enable automated single cellcapture and any or all of associated processing of the captured cells.In more detail, the user can be removed from part or all of the method(e.g. loading samples, capping lids, on-instrument lysis, reversetranscription processes, cDNA amplification, bead or cDNA productretrieval, on-instrument library preparation and cleanup, etc.). Thesystem can additionally or alternatively function to enhance theaccuracy (e.g. by minimizing manual processes) of cell capture andsample processing protocols. Additionally, through use of limited-useand/or pre-loaded reagent cartridges, the system 100 can provide astreamlined user experience with optimized quality control and designarchitecture to accommodate on-going development of assays and futureapplications. As such, the system confers the benefit of independent ornearly independent control of reagents or reagent groups. In a specificexample of this variation, the system includes a reagent cassette havingany or all of the following dedicated regions: a room temperatureregion, a cooling region, a heating/thermo-cycling region, a magneticregion (e.g., overlapping with a heating region), a region to provide acell sample input, a region for prepared library output or any othersuitable region. In a related benefit, the system and/or method canconfer the benefit of enabling the user to purchase smaller volumes ofreagents, such as through the distribution of reagents inprotocol-specific types and quantities to be used in accordance withspecific automated protocols. This can function to save costs, reducereagent waste, or have any other suitable outcome.

Additionally, through use of fluid handling and separation elements(e.g., magnetic separation components), embodiments of the system 100can function to provide automated sample and library cleanup steps.Relatedly, the system loo can confer the benefit of establishing betterfluid flow throughout the system. In a first example, this is enabledthrough an automated pipetting system (e.g., pipettor, gantry, andassorted pipette tips), which can monitor and/or direct fluid flow(e.g., to maintain an optimal flow rate, to establish an optimal volumeof reagents, etc.) without or with minimal user intervention.

Additionally, the system 100 can enable low parameter flow applications,high parameter flow applications, mass cytometry applications,proteogenomic applications, single cell RNA applications, proteindetection applications, and other applications, by allowing standardusers with various skill levels (e.g., novices, experts) to operateplatform components. Furthermore, in relation to performance, the systemloo can process cells or other biological material to rapidly generatepurified libraries, perform next generation sequencing (NGS)preparation, and perform other processes in a streamlined process (e.g.,by a set of dedicated consumables including an efficiently loadedreagent cartridge, a sample processing cartridge, and a container offluid handling disposables).

In specific embodiments, the system 100 can comply with use requirementsincluding one or more of: providing automated processes for nucleic acidlibrary preparation, ability to provide quality control at desiredpoints of a run, providing complete and single use kits for variousassays, providing validated and locked protocols, providing alignmentand retention of various system components, providing means formonitoring and controlling system operation (e.g., with a touchdisplay), providing remote monitoring capabilities, providing sampleprocessing within 24 hours, providing visual and/or audible systemnotifications, providing the ability to be cleaned with standardlaboratory cleaners and without disassembly, fitting on a standardlaboratory bench, providing easy installation, providing assay materialswith stable shelf life, returning reports of maintenance history,providing data storage (e.g., in relation to external storage media, inrelation to cloud storage, etc.), providing training, and providingother suitable functions according to various requirements.

As described above, in relation to sample processing, embodiments of thesystem 100 can include or be configured to process cells, cell-derivedmaterial, and/or other biological material (e.g., cell-free nucleicacids). The cells can include any or all of mammalian cells (e.g., humancells, mouse cells, etc.), embryos, stem cells, plant cells, or anyother suitable kind of cells. The cells can contain target material(e.g., target lysate, mRNA, RNA, DNA, etc.) which originates within thecells and is optionally captured by the cell capture system forprocessing. Additionally, the containers containing the cells can beprepared from multiple cell-containing samples (e.g., 12 samples, 24samples, 48 samples, 96 samples, 384 samples, 1536 samples, othernumbers of samples), wherein the various samples are hashed or barcodedprior to mixing them together into a single container (or reduced numberof containers). This feature enables automated processing of multiplesamples in the same automated run for their respective single cellpreparation and library preparation operations. Additionally oralternatively, the system 100 can be configured to interact withparticles (e.g., beads, probes, nucleotides, oligonucleotides,polynucleotides, etc.), droplets, encapsulated cells, encapsulatedbiomarkers, reagents, or any other suitable materials.

The system can further additionally or alternatively include any or allof the system components as described in U.S. application No.16/048,104, filed 27 Jul. 2018; U.S. application No. 16/049,057, filed30 Jul. 2018; U.S. application Ser. No. 15/720,194, filed 29 Sep. 2017;U.S. application Ser. No. 15/430,833, filed 13 Feb. 2017; U.S.application Ser. No. 15/821,329, filed 22 Nov. 2017; U.S. applicationSer. No. 15/782,270, filed 12 Oct. 2017; U.S. application Ser. No.16/049,240, filed 30 Jul. 2018; U.S. application Ser. No. 15/815,532,filed 16 Nov. 2017; U.S. application Ser. No. 16/115,370, filed 28Aug.2018, U.S. application Ser. No. 16/564,375, filed 9 Sep. 2019, and U.S.application Ser. No. 16/816,817, filed 12 Mar. 2020, which are eachincorporated in their entirety by this reference.

2.1 System: Deck

As shown in FIGS. 1A-1D, the deck 110 functions as a platform to supportand position one or more components of the system 100 (e.g., at a topbroad surface, at a top and bottom broad surface, at a side surface,etc.) for automated sample processing. Furthermore, the deck 110 canfunction to position one or more components of the system 100 to alignwith or otherwise interact with fluid processing subsystems, heatingsubsystems, separation subsystems (e.g., magnetic separationsubsystems), and/or other subsystems coupled to the gantry 170 and/orbase 180, as described below. In this regard, the deck 110 can bestationary as a reference platform, while other components are actuatedinto position for interacting with elements of the deck 110.Alternatively, the deck 110 can be coupled to one or more actuators forpositioning elements of the deck 110 for interactions with othersubsystems.

In the embodiment shown in FIGS. 1A-1D, the deck 110 provides a platformsupporting the set of sample processing elements, where the sampleprocessing elements can include disposable and/or reusable components,where the components include containers for containing sample processingmaterials and/or tools for processing samples (e.g., in relation tofluid handling, in relation to material separation, in relation toheating and cooling, etc.). In embodiments, the deck 110 can support aset of sample processing elements including one or more units of: areagent cartridge 120, a sample processing cartridge 130, a toolcontainer 140, a heating and cooling subsystem 150, a pumping subsystem157, a fluid level detection subsystem 159, and a separation subsystem160. Additionally or alternatively, the deck 110 can include othersuitable components (e.g., fluorescence detection subsystems, confocalmicroscope subsystems, spectroscopic detection subsystems, TotalInternal Reflection Fluorescence (TIRF) subsystems, Nuclear MagneticResonance (NMR) subsystems, Raman Spectroscopy (RS) RS subsystems,etc.).

The sample processing elements can be supported in a co-planar manner bythe deck 110, or alternatively at different planes. Preferably, discreteelements supported by the deck are non-overlapping, but alternativeembodiments of the deck 110 can support the sample processing elementsin an overlapping manner (e.g., for conservation of space, etc., foroperational efficiency, etc.).

As shown in FIGS. 1A and 1D, the deck 110 can be accessible by door 90,where the door 90 of the system 100 can transition between open and/orclosed modes in order to provide access to the deck 110 and elementssupported by the deck 110. However, in other variations, the deck 110may not be accessible by door 90.

Details of embodiments, variations, and examples of elements supportedby the deck 110 are further described in Sections 2.1.1 through 2.1.5below.

2.1.1 Deck-Supported Element: Reagent cartridge

The deck 110 includes at least one region 111 (shown in FIGS. 2A and 2B)for supporting a unit of the reagent cartridge 120, where the region 111functions to position the reagent cartridge 120 relative to portions ofthe heating and cooling subsystem 150, and separation subsystem 160described in more detail below. In this regard, the region 111 caninclude one or more openings, recesses, and/or protrusions for providinginterfaces between complementary portions of the reagent cartridge 120and associated portions of the heating and cooling subsystem 150 andseparation subsystem 160, and additionally to promote and maintainalignment between such portions.

The reagent cartridge 120 functions to contain, in one or morecompartments, materials for cell capture and/or processing of samplesaccording to one or more workflows for various applications. As such,the reagent cartridge 120 can define a set of storage volumesdistributed across a set of domains, where the set of domains can beconfigured for providing suitable environments for the material contentsof each domain. The set of storage volumes can directly contain sampleprocessing materials, and/or can alternatively be configured to receiveand maintain positions of individual containers (e.g., tubes, etc.) thatcontain sample processing materials. The storage volumes of each domaincan be distributed in arrays, or otherwise arranged. Storage volumes canhave circular cross sections, rectangular cross sections, or othermorphologies (e.g., cross sections, widths, depths, etc.) depending uponapplication of use (e.g., cold storage, heat transfer, magneticseparation, etc.).

The set of domains can additionally or alternatively be configured toprovide modularity, where one or more domains can be pre-packaged withmaterials that are stable over longer shelf lives, while other domainscan be configured to receive materials that have short shelf lives(e.g., immediately prior to use). The set of domains can additionally oralternatively be configured to promote operational efficiency (e.g., inrelation to grouping similar materials, etc.) for apparatuses thatinteract with materials of the reagent cartridge 120. The set of domainscan additionally or alternatively define regions for receiving and/orprocessing material (e.g., nucleic acid material) extracted from thesample processing cartridge 130 described in more detail below.

Additionally or alternatively, domains of the set of domains can beseparate (e.g. domain for receiving heat is separate from domains thatare intended for other storage temperatures or applications requiringdifferent temperatures), overlapping, or otherwise arranged. Domains ofthe set of domains can additionally or alternatively be distinguishedfrom each other by a morphology (e.g., length of the storage volumes ofeach domain, depth of storage volumes for accessing or interfacing withother elements of the deck, width or depth of domains configured forefficient heat transfer, etc.). In some variations, the set of domainscan further include at least one domain supporting an absorbent orporous material pad that can be used for receiving drips of fluid (e.g.,from a tip of a pipettor, described below) during processing. Theinternal surface properties for certain domains (e.g., for PCRreactions, for magnetic separation, etc.) may be configured with highsurface polish to enable low binding or retention of biomolecules (e.g.,nucleic acids or proteins). The various domains may also be mixed andmatched to provide a large number of available assays to the customers.

Individual storage volumes of the set of storage volumes of the reagentcartridge 120 can further include one or more seals, which function toisolate materials within the reagent cartridge 120, to preventcross-contamination between materials within individual storage volumes,to prevent contaminants from entering individual storage volumes, and/orto prevent evaporative loss during storage and shipment. The seal(s) canbe puncturable seal(s) (e.g., composed of paper, composed of a metalfoil, and/or composed of any other suitable material). However, theseal(s) can alternatively be configured to be non-puncturable (e.g., theseal(s) can be configured to peel away from the reagent cartridge 120).In embodiments, certain reagent containers may also be sealed by ahinged lid that can be opened or closed by a tool (e.g., as described inmore detail below), as needed for processing at appropriate steps of theprotocol.

In variations, the set of domains can include a first domain for storingreagents requiring a chilled environment (e.g., at a temperature from1C-15C), a second domain for storing materials that can be stored inambient conditions, a third domain storing tubes with materials forperforming polymerase chain reaction (PCR) operations and interfacingwith heating elements described below, a fourth domain for storingfunctionalized particles (e.g., beads with probes having barcodingregions and other functional regions, as described in U.S. applicationSer. No. 16/115,370, etc.), and a fifth domain for performing separationoperations (e.g., separation of target from non-target material bymagnetic force). In variations, domains providing different environmentsfor the storage volumes can be configured differently. For instance, thefirst domain (i.e., for cold storage) can be composed of a thermallyinsulating material and/or can include insulating material about storagevolumes of the domain (e.g., individually, about the entire domain).Additionally or alternatively, a domain for separation can be includemagnetically conductive materials configured to provide proper magneticfield characteristics for separation. Additionally or alternatively,domains for thermocycling or other heat transfer applications can beconfigured with thermally conductive materials to promote efficient heattransfer to and from the reagent cartridge. In embodiments, variousdomains can be optimally positioned such that there is minimalcross-talk between certain operations. For example, the domain(s) forchilled reagent storage volumes can be maintained a temperature (e.g.,4C) during a run, whereas the domain(s) for PCR reactions can requireheating (e.g., up to 95C during denature). As such, to minimize theeffect of PCR thermocycling on chilled reagents, the domain(s)containing the reagents stored at ambient temperature may be configuredin between the PCR thermocycling domain(s) and chilled domain(s). Inorder to further prevent heat cross-talk, additional buffer tubes withjust air may be used in between critical domains that need independenttemperature control.

In variations, process materials supported by the domains of the reagentcartridge 120 can include one or more of: buffers (e.g. ethanol, primingbuffer, lysis buffer, custom lysis buffers, sample wash buffers, salinewith RNAase inhibitors, bead wash buffers, RT buffer, buffer, etc.),oils (e.g. perfluorinert oil), PCR master mixtures, cells, beads (e.g.functionalized beads) or any other suitable materials used for cellcapture and/or sample processing. Additionally or alternatively, one ormore of the set of storage volumes can be empty (e.g. initially empty,empty throughout one or more processes, empty prior to filling by anoperator, etc.). Different storage regions in various domains of thereagent cartridge can have initial reagent volumes from a fewmicroliters (e.g., 5 microliters) to 50 milliliters. Additional detailsof process materials and applications of use are described below inrelation to workflows of Section 3.

In a specific example, as shown in FIG. 3 A, the reagent cartridge 120′includes a first domain 121′ at a first peripheral region of the reagentcartridge 120′ for storing reagents requiring a chilled environment, asecond domain 122′ at a central region of the reagent cartridge 120′ forstoring materials that can be stored in ambient conditions, a thirddomain 123′ at a peripheral region of the cartridge, near the seconddomain 122, for storing tubes with materials for performing polymerasechain reaction (PCR) operations, a fourth domain 124′ at a peripheralregion of the reagent cartridge 120′, for storing functionalizedparticles (e.g., beads with probes having barcoding regions and otherfunctional regions, as described in U.S. application Ser. No.16/115,370, etc.), and a fifth domain 125′ at a peripheral region of thereagent cartridge 120′ for performing separation operations (e.g.,separation of target from non-target material by magnetic force). In thespecific example, the fourth domain 124′ can be a modular element,whereby the fourth domain 124′ can be stored separately from the rest ofthe reagent cartridge 120′ until the functionalized particles are readyfor use, at which point the fourth domain 124′ is set in position andcoupled with the reagent cartridge 120′.

In the specific example, the first domain 121′ and the second domain122′ are covered by a first seal composed of a metal foil, the thirddomain 123′ and the fifth domain 125′ are covered by a second sealcomposed of a paper, and the fourth domain 124′ is covered by a thirdseal composed of a metal foil. However, variations of the example of thereagent cartridge 120′ can be configured in another suitable manner.

In another specific example for a 3.1. RNA processing protocol (e.g.,corresponding to the workflow of Section 3.1. below) shown in FIG. 3B,the reagent cartridge 120″ can include: a first storage volume 1201(e.g., having a volume of 4.46 mL) for 100% molecular grade ethanol; asecond storage volume 1202 (e.g., having a volume of 6.6 mL) for a firstwash buffer; a third storage volume 1203 (e.g., having a volume of 1.1mL) for a particle binding buffer; a fourth storage volume 1204 (e.g.,having a volume of 1.1 mL) for a lysis buffer; a fifth storage volume1205 (e.g., having a volume of 1.1 mL) for perfluorinert oil; a sixthstorage volume 1206 (e.g., having a volume of 6.63 mL) for a particlebinding wash solution; a seventh storage volume 1207 (e.g., having avolume of 1.1 mL) for a pre RT reaction wash buffer; an eighth storagevolume 1208 (e.g., having a volume of 1 mL) for a 0.1M sodium hydroxidesolution; a ninth storage volume 1209 (e.g., having a volume of 2.5 mL)for a second wash buffer; a tenth storage volume 1210 (e.g., having avolume of 1 mL) for mineral oil; an 11^(th) storage volume 1211 (e.g.,having a volume of 1.1 mL) for 80% molecular grade ethanol; a 12^(th)storage volume 1212 (e.g., having a volume of 2.2 mL) for nuclease-freewater; a 13^(th) storage volume 1213 (e.g., having a volume of 12.36 mL)for waste; a 14^(th) storage volume 1214 (e.g., having a volume of 0.15mL) for 0.1M DTT; a 15^(th) storage volume 1215 for an RT cocktailwithout superscript IV: a 16^(th) storage volume 1216 (e.g., having avolume of 0.011 mL) for superscript IV enzyme; a 17^(th) storage volume1217 (e.g., having a volume of 0.22 mL) for exonuclease buffer; an18^(th) storage volume 1218 (e.g., having a volume of 0.022 mL) forexonuclease enzyme; a 19^(th) storage volume 1219 (e.g., having a volumeof 0.128 mL) for a second strand synthesis mixture; a loth storagevolume 1220 (e.g., having a volume of 0.072 mL) for a second strandsynthesis primer; a 21^(st) storage volume 1221 (e.g., having a volumeof 0.22 mL) for a PCR master mixture for mRNA amplification; a 22^(nd)storage volume 1222 (e.g., having a volume of 0.33 mL) for a mixture(e.g., Kapa Biosystems™ HiFi HotStart Ready Mixture (2×); a 23^(rd)storage volume 1223 (e.g., having a volume of 0.025 mL) for mRNAproduct; a 24^(th) storage volume 1224 (e.g., having a volume of 0.11mL) for functionalized particles; a 25^(th) storage volume 1225 (e.g.,having a volume of 0.03 mL) for magnetic retrieval particles; a 26^(th)storage volume 1226 (e.g., having a volume of 0.66 mL) for AMPure XPparticles; 27^(th) storage volume 1227 for magnetic particle retrievalcollection; a 28^(th) storage volume 1228 for magnetic particlepreparation; a 29^(th) storage volume 1229 for magnetic particle secondstrand synthesis; a 30^(th) storage volume 1230 for magnetic particleretrieval post-PCR cDNA amplification; a 31^(st) storage volume 1231 forAMPure XP particle purification; a 32^(nd) storage volume 1232 for afirst exonuclease treatment; a 33^(rd) storage volume 1233 for a secondexonuclease treatment; a 34^(th) storage volume 1234 for second strandsynthesis; a 35^(th) storage volume 1235 for a first cDNA amplificationoperation; a 36^(th) storage volume 1236 for a second cDNA amplificationoperation; a 37^(th) storage volume 1237 for a third cDNA amplificationoperation; a 38^(th) storage volume 1238 for a fourth cDNA amplificationoperation; and a 39^(th) storage volume 1239 for a cell suspension.

In another specific example for a CITE-Seq processing protocol (e.g.,corresponding to workflow in Section 3.3 below) shown in FIG. 3C, thereagent cartridge 120′″ can include: a first storage volume 1201′ (e.g.,having a volume of 4.46 mL) for 100% molecular grade ethanol; a secondstorage volume 1202′ (e.g., having a volume of 6.6 mL) for a first washbuffer; a third storage volume 1203′ (e.g., having a volume of 1.1 mL)for a particle binding buffer; a fourth storage volume 1204′ (e.g.,having a volume of 1.1 mL) for a lysis buffer; a fifth storage volume1205′ (e.g., having a volume of 1.1 mL) for perfluorinert oil; a sixthstorage volume 1206′ (e.g., having a volume of 6.63 mL) for a particlebinding wash solution; a seventh storage volume 1207′ (e.g., having avolume of 1.1 mL) for a pre RT reaction wash buffer; an eighth storagevolume 1208′ (e.g., having a volume of 1 mL) for a 0.1M sodium hydroxidesolution; a ninth storage volume 1209′ (e.g., having a volume of 2.5 mL)for a second wash buffer; a tenth storage volume 1210′ (e.g., having avolume of 1 mL) for mineral oil; an 11^(th) storage volume 1211′ (e.g.,having a volume of 1.1 mL) for 80% molecular grade ethanol; a 12^(th)storage volume 1212′ (e.g., having a volume of 2.2 mL) for nuclease-freewater; a 13^(th) storage volume 1213′ (e.g., having a volume of 12.36mL) for waste; a 14^(th) storage volume 1214′ (e.g., having a volume of0.15 mL) for 0.1M DTT; a 15^(th) storage volume 1215′ for an RT cocktailwithout superscript IV: a 16^(th) storage volume 1216′ (e.g., having avolume of 0.011 mL) for superscript IV enzyme; a 17^(th) storage volume1217′ (e.g., having a volume of 0.22 mL) for exonuclease buffer; an18^(th) storage volume 1218′ (e.g., having a volume of 0.022 mL) forexonuclease enzyme; a 19^(th) storage volume 1219′ (e.g., having avolume of 0.128 mL) for a second strand synthesis mixture; a 20^(th)storage volume 1220′ (e.g., having a volume of 0.072 mL) for a secondstrand synthesis primer; a 21^(st) storage volume 1221′ (e.g., having avolume of 0.22 mL) for a PCR master mixture for cDNA amplification; a22^(nd) storage volume 1222′ (e.g., having a volume of 0.33 mL) for amixture (e.g., Kapa Biosystems™ HiFi HotStart Ready Mixture (2×); a23^(rd) storage volume 1223′ for an indexing primer; a 24^(th) storagevolume 1224′ (e.g., having a volume of 0.11 mL) for a PCR master mixturefor mRNA amplification; a 25 ^(th) storage volume 1225′ (e.g., having avolume of 0.2 mL) for ADT product; a 26^(th) storage volume 1226′ (e.g.,having a volume of 0. 025 mL) for mRNA product; a 27^(th) storage volume1227′ (e.g., having a volume of 0.11 mL) for functionalized particles; a28^(th) storage volume 1228′ (e.g., having a volume of 0.03 mL) formagnetic retrieval particles; a 29^(th) storage volume 1229′ (e.g.,having a volume of 0.66 mL) for AMPure XP particles; 30^(th) storagevolume 1230′ for magnetic particle retrieval collection; a 31^(st)storage volume 1231′ for magnetic particle preparation; a 32^(nd)storage volume 1232′ for magnetic particle second strand synthesis; a33^(rd) storage volume 1233′ for magnetic particle retrieval post-PCRcDNA amplification; a 34^(th) storage volume 1234′ for AMPure XPparticle purification; a 35^(th) storage volume 1235′ for a firstantibody-derived tag purification operation; a 36^(th) storage volume1236′ for a second antibody-derived tag purification operation; a37^(th) storage volume 1237′ for AMPure XP particle ADT post-PCRpurification; a 38^(th) storage volume 1238′ for mRNA AMPure XP particlepurification; a 39^(th) storage volume 1239′ for mRNA AMPure XPparticles post-PCR purification; a 40^(th) storage volume 1240′ for afirst exonuclease treatment; a 41st storage volume 1241′ for a secondexonuclease treatment; a 42^(nd) storage volume 1242′ for second strandsynthesis; a 43^(rd) storage volume 1243′ for a first cDNA amplificationoperation; a 44^(th) storage volume 1244′ for a second cDNAamplification operation; a 45^(th) storage volume 1245′ for a third cDNAamplification operation; a 46^(th) storage volume 1246′ for a fourthcDNA amplification operation; a 47^(th) storage volume 1247′ forantibody-derived tag fraction amplification; a 48^(th) storage volume1248′ for mRNA amplification; and a 49^(th) storage volume 1249′ for acell suspension.

2.1.2 Deck-Supported Element: Sample cartridge

As shown in FIGS. 2A and 2B, the deck 110 also includes at least oneregion 112 for supporting a unit of the sample processing cartridge 130,where the region 112 functions to position the sample processingcartridge 130 relative to portions of the heating and cooling subsystem150, the pumping subsystem 157, and the fluid level detection subsystem159 described in more detail below. In this regard, the region 112 caninclude one or more openings, recesses, and/or protrusions for providinginterfaces between complementary portions of the sample processingcartridge 130 and associated portions of the heating and coolingsubsystem 150, the pumping subsystem 157, and the fluid level detectionsubsystem 159, and additionally to promote and maintain alignmentbetween such portions.

The sample processing cartridge 130 functions to provide one or moresample processing regions in which cells are captured and optionallysorted, processed, or otherwise treated for downstream applications,where the downstream applications can be performed on the sampleprocessing cartridge 130 (e.g., on-chip) and/or away from the sampleprocessing cartridge 130 (e.g., off-chip). Portions of the sampleprocessing cartridge 130 can be configured within a single substrate,but can additionally or alternatively include multiple portions (e.g.connected by fluidic pathways) across multiple substrates.

As shown in FIGS. 4A-4C, an example of the sample processing cartridge130′ can include a base substrate 131 to which other elements arecoupled and/or in which other elements are defined. Furthermore, inrelation to sample processing involving microfluidic elements, the basesubstrate 131 can function as a manifold for fluid transfer tomicrofluidic elements, accessing of sample processing volumes at variousstages of processing, and transfer of waste materials produced duringsample processing. In variations, the base substrate 131 supports one ormore of: a sample processing chip 132, an inlet reservoir 133 forreceiving sample material (e.g., containing cells, containing particles,etc.) and delivering it into the sample processing chip 132, an accessregion 134 for accessing one or more regions of the sample processingchip 132, a lid 135 covering the access region and including a gasket136 providing sealing functions, and a waste containment region 137 forreceiving waste material from the sample processing chip 132. Thecartridge may have additional gasketed ports to also connect withoff-cartridge pumping system present in the instrument. Variations ofthe base substrate 131 can, however, include other elements. Forinstance, as described in more detail below, the base substrate caninclude one or more openings, recesses, and/or protrusions that providefurther coupling with the sample processing chip 132, in order tocollectively define valve regions for opening and closing flow throughthe sample processing chip 132.

As shown in FIGS. 4A and 4C (bottom view), the sample processing chip132, (equivalently referred to herein as a microwell device or a slide)defines a set of wells (e.g. microwells). Each of the set of wells canbe configured to capture a single cell and/or one or more particles(e.g., probes, beads, etc.), any suitable reagents, multiple cells, orany other materials. In variations, microwells of the sample processingchip 132 can be configured for co-capture of a single cell with a singlefunctional particle, in order to enable analyses of single cells and/ormaterials from single cells without contamination across wells.Embodiments, variations, and examples of the sample processing chip 132are described in one or more of: U.S. application Ser. No. 16/048,104,filed 27 Jul. 2018; U.S. application Ser. No. 16/049,057, filed 30 Jul.2018; U.S. application Ser. No. 15/720,194, filed 29 Sep. 2017; U.S.application Ser. No. 15/430,833, filed 13 Feb. 2017; U.S. applicationSer. No. 15/821,329, filed 22 Nov. 2017; U.S. application Ser. No.15/782,270, filed 12 Oct. 2017; U.S. application Ser. No. 16/049,240,filed 30 Jul. 2018; U.S. application Ser. No. 15/815,532, filed 16 Nov.2017; U.S. application Ser. No. 16/115,370, filed 28 Aug. 2018, U.S.application Ser. No. 16/564,375, filed 9 Sep. 2019, and U.S. applicationSer. No. 16/816,817, filed 12 Mar. 2020, which are each incorporated intheir entirety by reference above.

In material composition, the sample processing chip 132 can be composedof microfabricated silicon or glass-fused silica materials, whichfunction to enable higher resolution of the set of wells, enabled, forinstance, by defining sharper edges (e.g., thinner well walls, wellwalls arranged at an angle approaching 90 degrees, etc.) in the set ofwells. Materials and fabrication processes described can further enableone or more smaller characteristic dimensions (e.g., length, width,overall footprint, etc.) of the microwell cartridge as compared toconventional chip designs. Additionally or alternatively, the substrateinclude any other suitable material, such as—but not limited to—apolymer, metal, biological material, or any other material orcombination of materials. Sample processing chip 132 may be fabricatedby various processes such as precision injection molding, precisionembossing, microlithographic etching, LIGA based etching, or by othersuitable techniques.

In some variations, one or more surfaces of the set of wells (e.g.,bottom surface, side surface, bottom and side surfaces, all surfaces,etc.) can be reacted with oligonucleotide molecules for capture ofbiomarkers from individual cells into individual microwells. Theoligonucleotide molecules present on each and individual microwells maybe barcoded to allow biomarkers processed in each microwell to be linkedback to a particular well and hence a particular single cell. In onevariation, the set of wells includes a set of microwells havinghexagonal cross sections taken transverse to longitudinal axes of thewells, as described in one or more of the applications incorporated byreference above.

In one variation, as shown in FIG. 4C, the sample processing chip 132can include an inlet opening 32, a first fluid distribution network 33downstream of the inlet opening, for distribution of fluids to a set ofmicrowells 34, a second fluid distribution network 35 downstream of theset of microwells 34, and an outlet opening 36 coupled to a terminalportion of the second fluid distribution network 35, for transfer ofwaste fluids from the sample processing chip 132. In this variation, thesample processing chip 132 is coupled to a first side (e.g., under-side)of the base substrate 131 (e.g., by laser welding, glue bonding, solventbonding, ultrasonic welding or another technique). Coupling of thesample processing chip 132 to the side of the base substrate 131 canenable transfer of heat from the heating and cooling subsystem 150 tothe set of microwells 34 and/or other regions of the sample processingchip 132, where the heating and cooling subsystem 150 is described inmore detail below.

The base substrate 131, as described above, can also include an inletreservoir 133 (e.g., defined at a second side of the base substrate 131opposing the first side to which the sample processing chip 132 iscoupled). The inlet reservoir functions to receive sample material(e.g., samples containing cells, sample containing barcoded cells,sample containing encapsulated materials, samples containing particles,etc.) and/or sample processing materials from the reagent cartridge 120described above, for delivery into the inlet opening 32 of the sampleprocessing chip 132. In variations, the inlet reservoir 133 can bedefined as a recessed region within a surface of the base substrate 131,wherein the recessed region includes an aperture that aligns with and/orseals with the inlet opening 32 of the sample processing chip 132. Theinlet reservoir 133 of the base substrate 131 can interface withupstream fluid containing components and/or bubble mitigatingcomponents, as described in one or more of: U.S. application Ser. No.16/048,104, filed 27 Jul. 2018; U.S. application Ser. No. 16/049,057,filed 30 Jul. 2018; U.S. application Ser. No. 15/720,194, filed 29 Sep.2017; U.S. application Ser. No. 15/430,833, filed 13 Feb. 2017; U.S.application Ser. No. 15/821,329, filed 22 Nov. 2017; U.S. applicationSer. No. 15/782,270, filed 12 Oct. 2017; U.S. application Ser. No.16/049,240, filed 30 Jul. 2018; U.S. application Ser. No. 15/815,532,filed 16 Nov. 2017; U.S. application Ser. No. 16/115,370, filed 28 Aug.2018, U.S. application Ser. No. 16/564,375, filed 9 Sep. 2019, and U.S.application Ser. No. 16/816,817, filed 12 Mar. 2020, which are eachincorporated in their entirety by reference above.

The inlet reservoir 133 can also be configured to interface with a fluidlevel detection subsystem 159 supported by or otherwise interfacing withthe deck 110, as described in more detail below. In particular, portionsof the inlet reservoir 133 can be composed of materials that enablesensing of fluid levels within the inlet reservoir 133 (e.g., by opticalinterrogation, by pressure sensing, by weight sensing, etc.). Forinstance, the inlet reservoir 133 can be composed of an opticallytransparent or translucent material to visible spectrum electromagneticradiation and/or non-visible spectrum electromagnetic radiation (e.g.,by fabrication with different materials, by fabrication to produce thinregions of material at the inlet reservoir 133, etc.), where sensingelements of the fluid level detection subsystem 159 can be configured tointerrogate a level of fluid within the inlet reservoir 133 accordingly.

In variations, one or more of the inlet reservoir 133 of the basesubstrate 131 and the inlet 32 of the sample processing chip 132 caninclude valve components that can be open or closed by one or morecomponents of the system 100. In a first variation, the inlet reservoir132 includes an aperture that can be accessed by a pipette tip or anyother suitable attachment of a fluid handling subsystem coupled to thegantry 170 (described in more detail below). In some embodiments, theaperture can be closed and therefore prevent fluid from traveling fromthe inlet reservoir 132 to the sample processing chip 132. The inletreservoir 132 can, however, be configured in another suitable manner.The opening associated with the inlet reservoir 133 may have a conicalshape surface open towards the top allowing interfacing and sealing apipette tip such that fluid (aqueous solutions or oil or air) may bepumped directly into the microchannel defined in 33 in FIG. 4C.

As shown in FIGS. 4A and 4B, the base substrate 131 can also define anaccess region 134 for accessing one or more regions of the sampleprocessing chip 132, where the access region can allow regions of thesample processing chip 132 to be observed and/or extracted from thesample processing chip 132 at various phases of sample processing. Asshown in FIGS. 4A and 4B, the access region 134 can be defined as arecessed region within the base substrate 131, and include an opening 37aligned with the region of the sample processing chip 132 that includesthe set of microwells. The sample processing chip 132 may have as few asloo microwells to as many as 100 million microwells. As such, invariations wherein the microwell region is open to the environment(e.g., without a covering to seal the wells), the opening 37 of theaccess region 134 can function as a microwell to provide access tocontents of the microwells for observation and/or material extraction(e.g., by magnetic separation, as described in further detail below).The opening 37 can match a morphology and footprint of the microwellregion, and in a first variation, as shown in FIG. 4B, can be a squareopening. However, in other variations, the opening 37 can have anothersuitable morphology.

As shown in FIGS. 4A-4C, the base substrate 131 can include or otherwisecouple to a lid 135 covering the access region 134, where the lid 135can include a gasket 136 providing sealing functions, and where the lid135 functions to transition the access region 134 between open andclosed modes, thereby preventing evaporative sample loss and/orcontamination of contents of the sample processing chip 132 duringoperation. The lid 135 can additionally or alternatively function toprotect the contents of the microwells or other processing regions ofthe sample processing chip 132 from debris, enable a processing of thecontents of the sample processing chip 132 (e.g. by isolating regionsfrom the ambient environment), initiate the start of a protocol (e.g.,by opening to accept reagents from a pipettor), prevent usermanipulation of the sample processing chip 132 (e.g., by closing afterall necessary reagents have been added), define (e.g., with the lid 135)part or all of a fluid pathway, cavity, or reservoir (e.g. serve as thetop surface of a fluidic pathway between the inlet and the set ofmicrowells, serve as a boundary of a fluid pathway adjacent themicrowell region, serve as the top surface of a fluidic pathway betweenthe set of wells and the waste chamber, etc.), or perform any othersuitable function.

As shown in FIG. 4B, in at least one variation, the lid 135 can becomplementary in morphology to features of the access region 134, suchthat the lid 135 mates with the access region 134, while providing a gapwith the sample processing chip 132. Additionally, in variations (shownin FIG. 4B and 4C), the lid 135 can be substantially flush with the basesubstrate 131 at a top surface when the lid 135 is in the closedposition. However, the lid 135 can be morphologically configured inanother suitable manner.

In variations, a protrusion 38 of the lid 135 can interface with theopening 37 of the access region 134, thereby substantially preventingaccess to the opening 37 when the lid is in the closed position. Asshown in FIG. 4B, in some variations, the protrusion 38 can have a base(or other region) surrounded by a gasket 136, which functions to sealthe opening 37 of the access region 134 in the closed position of thelid 135. Variations of the lid 135 can, however, omit a gasket andpromote sealing of the access region 134 in another suitable manner.

In some variations, the lid 135 can include a locking or latchingmechanism that allows the lid 135 to be maintained in the closedposition with the base substrate 131 until the locking/latchingmechanism is released. In the variation shown in FIGS. 5A-5C, aperipheral portion of the lid 135 can include a one or more tabs 39 thatinterface with corresponding tab receiving portions of the basesubstrate 131, where, the tabs 39 are configured to flex when pushedinto the base substrate 131 until they interface with the tab receivingportions of the base substrate 131 and return from a flexedconfiguration to a latched state. Additionally or alternatively, in thevariation shown in FIGS. 5A-5C, the locking/latching mechanism caninclude a releasing body 41 (e.g., bar, recess, hook, etc.) that can beinterfaced with in order to release the tab(s) 39 from the tab receivingportions, and transition the lid 135 from the closed mode to the openmode in relation to the base substrate 131. As such, the lid 135provides the lid an open mode in which the access region 134 isuncovered and a closed mode in which the access region 134 is covered.In the variation shown in FIGS. 5A-5C, the releasing element 41 includesa bar that is recessed away from the access region 134 of the basesubstrate 131, where the bar can be reversibly coupled to a lid-openingtool 145. In variations, the lid-opening tool 145 can include a firstregion (e.g., first end) that interfaces with a an actuator (e.g.,actuating tip, pipettor of a fluid handling subsystem coupled to thegantry 170 described below, etc.), and a second region (e.g., secondend) including a linking element 42 configured to interface with thereleasing element 41 of the lid 135. Then, with movement of thepipettor/pipette interface, the lid-opening tool 145 can be configuredto pull on the releasing element 41 and/or push on the lid 135 in orderto transition the lid between open and/or closed modes. As such, inrelation to fluid handling elements coupled to the gantry 170 describedbelow, the system Dm can provide operation modes for: coupling alid-opening tool 145 to an actuator (e.g., coupled to a gantry 170), thelid-opening tool including a linking element 42; moving the lid-openingtool into alignment with a releasing element 41 of the lid 135,reversibly coupling the linking element 42 with the releasing element41; and applying a force to the releasing element 41, thereby releasingthe lid 135 from a latched state and transitioning the lid 135 from aclosed mode to an open mode. In order to effectively apply an unlatchingforce (e.g., by the actuator (e.g., coupled to a gantry 170), the basesubstrate 131 can be retained in position (e.g., by retention elementsdescribed in Section 2.1.4, by retention elements of the heating andcooling subsystems, by retention elements of the fluid level detectionsubsystem, by retention elements of the deck, etc.) which passively oractively apply counteracting forces against the unlatching forcesapplied through the lid-opening tool 145.

In variations, however, the locking/latching mechanism can additionallyor alternatively include or operate by way of: a lock-and-key mechanism,magnetic elements, or another suitable mechanism. Furthermore, inalternative variations, the lid 135 can include another lid actuator,for instance, including a motor that rotates the lid about an accessparallel to a broad surface of the sample processing cartridge 130. Theactuator can additionally or alternatively be configured to translatethe lid 135 (e.g. slide the lid 135 parallel to a broad surface of thesample processing cartridge 130, translate the lid 135 perpendicular tothe broad surface, etc.) or otherwise move the lid 135 to selectivelycover and uncover one or more predetermined regions (e.g. the set ofmicrowells). As such, the lid 135 can be configured to operate in anautomated or semi-automated fashion, such that the lid 135 automaticallycloses upon one or more triggers (e.g., cell capture protocol isinitiated by a user, cell processing protocol is initiated by a user,all reagents for a selected protocol have been added from the reagentcartridge 120, etc.) and opens upon one or more triggers (e.g., cellcapture protocol has been completed, upon user request, it has beendetermined that the cells are viable, it has been determined that singlecells have been captured, etc.). Additionally or alternatively,operation of the lid 135 can be initiated and/or completed by a user,operated according to a schedule or other temporal pattern, or otherwiseoperated.

As shown in FIGS. 4A-4C, the base substrate 131 can also include a wastecontainment region 137 for receiving waste material from the sampleprocessing chip 132. The waste containment region 137 can also functionto maintain desired pressures (e.g., vacuum pressures, etc.) within thesample processing chip 132, thereby enabling flow of liquid from theinlet reservoir 133 through the sample processing chip 132 and to thewaste containment region 137. The waste containment region 137 can bedefined as a volume (e.g., recessed into the base substrate 131,extending from the base substrate 132, coupled to an outlet of the basesubstrate 131, etc.) for receiving waste or other materials from thesample processing chip 132. In the variation shown in FIGS. 4A-4C, thewaste containment region 137 is defined at a side of the base substrate131 opposing the side to which the sample processing chip 132 iscoupled, such that waste from the sample processing chip 132 is pushedor pulled upward into the waste containment region 137 by forces of thepumping subsystem 157 described in more detail below. However, the wastecontainment region 137 can additionally or alternatively be configuredin another suitable position relative to the base substrate 131 and thesample processing chip 132, in order to receive waste.

The waste containment region 137 can have a volumetric capacity of10-100 mL or another suitable volumetric capacity.

As shown in FIGS. 4A-4C, the waste containment region 137 can include acover 48 (e.g., a cover that is approximately co-planar with the lid135), which facilitates containment of waste within the wastecontainment region 137. Alternatively, the waste containment region 137may not include a cover. Furthermore, as shown in FIG. 4C, examples ofthe waste containment region 137 can include a pump outlet 51 distinctfrom the cover, where the pump outlet 51 can allow the residual air inthe waste chamber to be pressurized by an off-cartridge pump(e.g., bypumping mechanisms, etc.); however, variations of the waste containmentregion 137 can alternatively omit a waste outlet.

In relation to the waste containment region 137, the system loo canfurther include a valve 43 configured to allow and/or prevent flow fromthe sample processing chip 132 to the waste containment region 137. Thevalve 43 can interface with the outlet opening 36 of the sampleprocessing chip 132 described above, in order to enable and/or blockflow out of the outlet opening 36 and into the waste containment region137. The valve 43 can have a normally open state and transition to aclosed state upon interacting with a valve-actuating mechanism.Alternatively, the valve 43 can have a normally closed state andtransition to an open state upon interacting with a valve-actuatingmechanism.

In the variation shown in FIGS. 4A and 6A-6B, the valve 43 comprises anelastomeric body and is configured to couple the sample processing chip132 to the base substrate 131 through an opening 44 of the sampleprocessing chip 132 that aligns with a corresponding valve-receivingportion of the base substrate 131. In this variation, a transitionableportion of the valve 43 is configured to be positioned along a flow pathfrom the outlet opening 36 of the sample processing chip 132 to theinlet of the waste containment region 137 of the base substrate 132(e.g., along a flow path from the microwell region to an outlet of thesample processing chip into a waste containment region of the sampleprocessing cartridge). In an example the opening 44 of the sampleprocessing chip 132 is contiguous with the outlet opening 37 of thesample processing chip 132; however, in other variations, the outletopening 37 and the opening 44 may be displaced from the each other andconnected by another microfluidic channel. As such, closure of the valve43 can block flow from the outlet opening 37 into the waste containmentregion 137, and the valve 43 can be opened to allow flow from the outletopening 37 into the waste containment region 137.

In a variation shown in the cross sectional images of the base substrate131 shown in FIG. 6B, a valve actuator 45 can access the base substrate131 from below (e.g., from below the deck), and pass through a channelor other recess/opening of the base substrate 132 in order to interactwith the valve 43. In particular, when a tip 46 (aligned with theopening into the base substrate) of the valve actuator 45 pushes againstthe valve (e.g., a elastomeric membrane of the valve 43), as shown inFIGS. 6B (top), the valve 43 can transition to a closed state in orderto fluidically decouple the outlet opening 37 of the sample processingchip 132 from the waste containment region 137. Additionally oralternatively, as shown in FIG. 6B (bottom), removal of force by thevalve actuator 45 can remove pressure from the valve 43 and transitionit to an open state to fluidically couple the outlet opening 36 of thesample processing chip 132 from the waste containment region 137. Assuch, the valve actuation subsystem includes an engaged mode wherein thetip extends into the valve opening to deform the elastomeric valve,thereby closing the flow path, and a disengaged mode wherein the tip isretracted, thereby opening the flow path. However, the valve 43 canadditionally or alternatively be configured in another suitable manner.

In other variations, the system can include a similar mechanism forcoupling a valve to other flow paths of the sample processing chip 132and/or to the base substrate 131.

Variations of the base substrate 131 can, however, include otherelements. For instance, as described in more detail below, the basesubstrate 131 can include one or more openings, recesses, and/orprotrusions that provide further coupling with the sample processingchip 132, in order to promote or inhibit flow through the sampleprocessing chip 132. For instance, as shown in FIG. 6A, the basesubstrate can include a pump opening 46 that couples the base substrate131 to a pumping element of the pumping subsystem 157 (e.g., throughdeck no), in order to drive and/or stop fluid flow through the sampleprocessing chip 132.

The base substrate 131 of the sample processing cartridge 130 can,however, include other suitable elements.

2.1.3 Deck-Supported Element: Tool Container

As shown in FIGS. 2A and 2B, the deck 110 includes at least one region113 for supporting a unit of the tool container 140, where the region113 functions to position the tool container 140 relative to fluidhandling apparatus of the gantry 170 described below. The region 113 canalso position the tool container 140 in proximity to the reagentcartridge 120 and sample processing cartridge 130 being used, in orderto provide a more compact system and improve efficiency of automatedoperations involving contents of one or more of the reagent cartridge120, sample processing cartridge 130, and tool container 140. The region113 can also include an opening that allows the tool container 140 to beat least partially recessed below a surface of the deck 110.

The tool container 140 functions to contain, in one or morecompartments, one or more units of various tools for fluid aspiration,fluid delivery, separation of target material from non-target materialof a sample, sample processing cartridge lid-opening tools 145, and/orother tools, according to one or more workflows for variousapplications. As such, the tool container 140 can facilitate transferand/or mixing of reagents with sample, fluidically couple and/ordecouple elements at various regions of the deck 110, or otherwiseinteract with one or more components of the system 100.

In variations, one of which is shown in FIG. 7A the tool container 140′can include a set of tips 141 for fluid aspiration and/or fluid delivery(e.g., to and from the reagent cartridge 120, to and from the sampleprocessing cartridge 130, etc.). The set of tips 141 can include any orall of: standard pipette tips (e.g. P20 tips, P200 tips, P1000 tips,etc.); additionally or alternatively, the set of tips can includepiercing tools (e.g., to gain access to a reagent tube through a seal),blunt tips (e.g. for facilitating fluid flow, for blocking an aperture,for defining a fluidic pathway, etc.), or any other suitable tips. Thetips can be configured or otherwise used in any or all of: piercing(e.g. piercing a reagent strip foil), transferring and/or mixing a setof reagents (e.g. a tip for mixing ethanol and priming buffer, a tip fortransferring SPRI ethanol, a tip for transferring SPRI supernatant, atip for transferring SPRI elution buffer, etc.), cell dispensing into aunit of the sample processing cartridge 130, dispensing offunctionalized particles into a unit of the sample processing cartridge,facilitating the performance of a predetermined set of processes (e.g.,particle washing, cell lysis, oil dispensing, pre-reverse-transcriptionwash, other workflows described below etc.), or can have any othersuitable function.

As shown in FIG. 7A, the set of tips 141 can be arranged in an array(e.g., according to type, according to size, etc.) within the toolcontainer 140, where the internal base of the tool container 140 caninclude drip-catching sections for catching drips leaving theaspiration/delivery ends of the tips from various fluid handlingoperations. Furthermore the tool container 140 can include spacingelements for preventing individual tips of the set of tips 141 fromcontacting each other, thereby preventing cross-contamination.Furthermore, regions of one or more of the tips for interfacing with thepipettor can be conductive (e.g., thermally conductive, electricallyconductive, composed of a metal, composed of a conductive polymer,composed of a semiconducting material, etc.).

In a specific example for a 3′ processing protocol shown in FIG. 7B, thetool container 140″ can include: a first tip 1401 (e.g., with a tipvolume of 200 mL, with a tip volume of 200 uL) for transferring ethanol,a wash buffer, and/or a priming solution; a second tip 1402 (e.g., witha tip volume of 1000 mL, with a tip volume of 1000 uL) for transferringof a cell suspension; a third tip 1403 (e.g., with a tip volume of 200mL, with a tip volume of 200 uL) for transferring functionalizedparticles; a fourth tip 1404 (e.g., with a tip volume of 1000 mL, with atip volume of 1000 uL) for transferring a wash buffer; a fifth tip 1405(e.g., with a tip volume of 1000 mL, with a tip volume of 1000 uL) fortransfer of a particle binding buffer; a sixth tip (e.g., with a tipvolume of 1000 mL, with a tip volume of 1000 uL) for transfer of DTIand/or a lysis buffer; a seventh tip 1407 (e.g., with a tip volume of1000 mL, with a tip volume of 1000 uL) for transfer of perfluorinertoil; an eighth tip 1408 (e.g., with a tip volume of 1000 mL, with a tipvolume of 1000 uL) for transfer of DTT, particle binding solution,and/or wash solution; a ninth tip 1409 (e.g., with a tip volume of 1000mL, with a tip volume of 1000 uL) for transfer of DTT and/or a pre-RTreaction wash solution; one or more magnetic sleeves 1410 for magneticretrieval; an 11th tip 1411 (e.g., with a tip volume of 1000 mL, with atip volume of 1000 uL) for transfer of DTT and/or an RT cocktailsolution; a 12^(th) tip 1412 (e.g., with a tip volume of 1000 mL, with atip volume of 1000 uL) for transfer of an exonuclease treatment; a13^(th) tip 1413 (e.g., with a tip volume of 1000 mL, with a tip volumeof 1000 uL) for transfer of mineral oil; a 14 ^(th) tip 1414 fortransfer of an exonuclease treatment; a 15^(th) tip 1415 (e.g., with atip volume of 1000 mL, with a tip volume of 1000 uL) for transfer of asodium hydroxide solution; a 16^(th) tip 1416 (e.g., with a tip volumeof 1000 mL, with a tip volume of 1000 uL) for transfer of a second washbuffer; a 17^(th) tip 1417 for transfer of a second strand synthesissolution; an 18^(th) tip 1418 (e.g., with a tip volume of 1000 mL, witha tip volume of 1000 uL) for transfer of a particle solution; a 19^(th)tip 1419 (e.g., with a tip volume of 1000 mL, with a tip volume of 1000uL)for transfer of a second wash buffer; a 20^(th) tip 1420 (e.g., witha tip volume of 1000 mL, with a tip volume of 1000 uL) for transfer of aPCR master mix and/or particle solution; a 21^(st) tip 1421 (e.g., witha tip volume of 1000 mL, with a tip volume of 1000 uL) for transfer ofmineral oil; a 22nd tip 1422 (e.g., with a tip volume of 1000 mL, with atip volume of 1000 uL) for transfer of a PCR product; a 23 ^(rd) tip1423 (e.g., with a tip volume of 1000 mL, with a tip volume of 1000 uL)for transfer of AMPure and/or supernatant; a 24^(th) tip 1424 (e.g.,with a tip volume of 1000 mL, with a tip volume of 1000 uL) for transferof ethanol; a 25^(th) tip 1425 (e.g., with a tip volume of 1000 mL, witha tip volume of 1000 uL) for transfer of ethanol; a 26^(th) tip 1426(e.g., with a tip volume of 50 mL, with a tip volume of 50 uL) fortransfer of ethanol; and a 27^(th) tip 1427 (e.g., with a tip volume of50 mL, with a tip volume of 50 uL) for transfer of nuclease-free waterand/or derived products.

In variations, the tool container 140 can additionally or alternativelyinclude other sample processing tools. In one variation, the toolcontainer 140 can include one or more units of a separation tool tip 142for magnetic separation of target material from non-target material(described in more detail below). Additionally or alternatively, invariations, the tool container 140 can additionally or alternativelyinclude units of a lid-opening tool 145 for transitioning the lid 135described above to an open configuration. However, in relation todisposability of the tool container 140 and/or its contents, the toolcontainer 140 can be configured to contain only disposable elements, andto omit reusable elements (e.g., units of a lid-opening tool 145, asshown in FIG. 2B).

Furthermore, units of contents of the tool container 140 canadditionally be included with one or more of the reagent cartridge 120,the sample processing cartridge 130, otherwise arranged at the deck 110,separate from the deck 110, separate from the system 100 or otherwisearranged. In other variations, re-useable tools used in the toolcontainer 140 may include other tools that use electrical,electromagnetic, optical or combination of different modalities tointeract with the gantry 17o and be moved to specific locations over thereagent cartridge 120 and/or the sample processing cartridge 130 toprovide specific energies (e.g., heat, optical signals, electromagneticwaves, etc.) and/or sense specific signals (e.g., optical, thermal,electromagnetic, etc). These tools may be wired to the controlelectronics or may be wirelessly charged and controlled/communicated.

2.1.4 Deck-Supported Elements—Registration and Retention Features

As shown in FIGS. 8A through 81 , the deck no can include a set ofretention elements positioned relative to region in for the reagentcartridge 120, region 112 for the sample processing cartridge 130, andregion 113 for the tool container 140, where the set of retentionelements function to register and retain the reagent cartridge 120,sample processing cartridge 130, and tool container 140 at the deck 110during operation to process samples, and to enable release of thereagent cartridge 120, sample processing cartridge 130, and toolcontainer 140 from the deck 110 when appropriate. The set of retentionelements can include mechanical retention elements (e.g., recesses,protrusions, etc.) configured provide retention by way of a snap fit, apress fit, ratcheting, or another suitable mechanism. Additionally oralternatively, the set of retention elements can include magneticretention elements or other suitable retention elements. In variationsincluding magnetic retention elements, associated magnets can bepositioned away from magnetic separation areas of the reagent cartridge120 and deck 110 (described in relation to 2.1.8 below) so as to notinterfere with associated magnetic separation mechanisms. Alternatively,the associated magnets can be positioned in proximity to the magneticseparation areas of the reagent cartridge 120 and deck 110, in order tofacilitate magnetic separation operations performed at the reagentcartridge 120 (and/or other suitable portions of the system.

The retention elements can provide uniform mechanisms for each of thereagent cartridge 120, sample processing cartridge 130, and toolcontainer 140. Alternatively, the reagent cartridge 120, sampleprocessing cartridge 130, and tool container 140 can each includedifferent retention elements that operate by different mechanisms asappropriate. The retention mechanisms supported by the set of retentionelements can be manually operated (e.g., a user interacts with theretention elements to disengage and/or engage a component with the deckno). Additionally or alternatively, the retention mechanisms supportedby the set of retention elements can be non-manually operated (e.g.,with actuators coupled to the retention elements in order to transitionthem between engaged and disengaged modes). Retention mechanisms areconfigured to operate with appropriate morphologies (e.g., to facilitateengagement by a manual operator or apparatus), loading and unloadingforces, and/or transmitted forces (e.g., to other sensitive elements ofthe deck 110).

In the variation shown in FIG. 8A, the deck 110 can include a firstsubset of retention elements 21 corresponding to region in for thereagent cartridge 120, a second subset of retention elements 22corresponding to region 112 for the sample processing cartridge 130, anda third subset of retention elements 23 corresponding to region 113 forthe tool container 140.

In relation to FIG. 8A, the first subset of retention elements 21 atregion 111 can include a set of snap ledges positioned about region 111(e.g., at contralateral sides of region in), in a manner thatcomplements corresponding retention interfaces 31 of the reagentcartridge 120. In more detail, as shown in FIGS. 8B and 8C, theretention interfaces 31 of the reagent cartridge 120′ can include a setof tabs (e.g., flexible tabs) that can be compressed from a baselineposition to be positioned at region in, where release from thecompressed configuration allows the set of tabs to latch with the snapledges. The set of tabs of the retention interfaces 31 can furtherinclude gripping features (e.g., bumps, etc.) that allow a user tocompress the set of tabs easily. In related variations, the first subsetof retention elements 21 and corresponding retention interfaces of thereagent cartridge 120′ can be positioned in another manner. Forinstance, the set of tabs of the reagent cartridge can be positioned ata non-peripheral region of the reagent cartridge 120 (e.g., with fingerand/or thumb hole cutouts), with corresponding alignment with theretention elements 21 of the deck 110. In relation to providing robustcontact, the retention elements 21 can be configured to provide abiasing force against deck elements (e.g., heating and cooling subsystemelements, magnetic separation subsystem elements, etc.) that the reagentcartridge 120 interacts with, in order to provide robust contact betweensurfaces. The biasing force used against the deck elements may be atleast 0.5 lbs or at least 1 lbs or at least 2 lbs, or at least 3 lbs.

In relation to FIG. 8A, the second subset of retention elements 22 atregion 112 can include a set of snap ledges positioned about region 112(e.g., at contralateral sides of region 112 and at a third peripheralside of region 112), in a manner that complements correspondingretention interfaces 32 of the sample processing cartridge 130. In moredetail, as shown in FIGS. 8D through 8G, the retention interfaces 32 ofthe sample processing cartridge 130 can include a set of tabs (e.g.,flexible tabs) that can be compressed from a baseline position to bepositioned at region 112, where release from the compressedconfiguration allows the set of tabs to latch with the snap ledges. Inthis variation, the sample processing cartridge 130 can be configured tobe inserted into/engaged with a first of the retention elements 22 a(e.g., at a side of the sample processing cartridge near the inletreservoir, at a side of the sample processing cartridge near the wastecontainment, region, etc.) prior to latching of the other retentioninterfaces 22 b with the contralateral snap ledges. Similar to the setof tabs of the reagent cartridge 120, the set of tabs of the retentioninterfaces 32 of the sample processing cartridge 130 can further includegripping features (e.g., bumps, etc.) that allow a user to compress theset of tabs easily. In relation to providing robust contact, theretention elements 22 can be configured to provide a biasing forceagainst deck elements (e.g., heating and cooling subsystem elements,pumping subsystem elements, etc.) that the sample processing cartridge130 interacts with, in order to provide robust contact between surfaces.Furthermore, the set of retention elements 22 of the deck no can beconfigured to reversibly engage the sample processing cartridge 120 andprovide a counteracting force against the lid-opening tool 145 describedin relation to lid-opening operation modes.

In relation to FIG. 8A, the third subset of retention elements 23 atregion 113 can include a set of snap ledges positioned about region 113(e.g., at contralateral sides of region 113), in a manner thatcomplements corresponding retention interfaces 33 of the tool container.In more detail, as shown in FIGS. 8H and 8I, the retention interfaces 33of the tool container 140 can include a set of tabs (e.g., flexibletabs) that can be compressed from a baseline position to be positionedat region 113, where release from the compressed configuration allowsthe set of tabs to latch with the snap ledges. The set of tabs of theretention interfaces 33 can further include gripping features (e.g.,bumps, etc.) that allow a user to compress the set of tabs easily.

In variations of the retention elements of the deck no described abovecan, however, include other suitable features or be configured relativeto associated elements in another suitable manner.

2.1.5 Deck-Supported Element: Heating and Cooling Subsystem

As shown in FIGS. 1A-1D and 2B, the system 100 can include a heating andcooling subsystem 150, which functions to transfer heat to and/or fromdesired regions of the reagent cartridge 120 and/or the sampleprocessing cartridge 130. The heating and cooling subsystem 150 canadditionally or alternatively function to maintain desired temperatureswithin internal volumes of the system 100. In variations, the heatingand cooling subsystem 150 can include one or more units of: heatingelements (e.g., Peltier heating elements, resistive heating elements,other heating elements), cooling elements (e.g., Peltier coolingelements, chilled aluminum block, fluidic pathway system to circulatecoolant, etc.), thermal contact or non-contact bodies for transferringheat to or from the heating and cooling elements to other objects, heatsinks, fans, temperature sensors, and thermal control circuitry (e.g.,with electrical coupling to processing elements of the base 180described in more detail below). In variations, the cooling element(s)can maintain storage volumes and/or samples between 2 and 8 degreesCelsius, further preferably at 4 degrees Celsius. Additionally oralternatively, the cooling elements can maintain one or more storagevolumes/samples at any suitable temperature (e.g. below 2 degreesCelsius, above 8 degrees Celsius, etc.).

One or more portions of the heating and cooling subsystem 150 can passinto openings of the deck no to thermally interface with or otherwisecouple with desired portions of other system elements (e.g., reagentcartridges, sample processing cartridges, tool container, etc.)supported by the deck 110, in order to provide heat transfer functionsfor various applications. Alternatively, the deck no can be composed ofa thermally conductive material at desired regions for heat transferapplications, and portions of the heating and cooling subsystem 150 canbe configured to contact the thermally conductive material regions ofthe deck 110 for heat transfer.

In the specific example shown in FIG. 2B, the heating and coolingsubsystem 150 includes a set of thermal bodies that thermally interfacethe heating elements with desired portions of the reagent cartridge 120and the sample processing cartridge 130 supported by the deck 110. Theset of thermal bodies includes a first thermal body 156′ (e.g., thermalplate) passing through an opening of the deck 110′ and configured tointerface with the first domain 121′ of the reagent cartridge 130′described above, where the first thermal body 156′ includes an array ofrecesses for surrounding storage volumes of the first domain 121′ (e.g.,to provide a chilled environment for materials stored in the storagevolumes of the first domain 121′). As shown in FIG. 2B, the set ofthermal bodies can also include a second thermal body 157′ (e.g.,thermal plate) passing through an opening of the deck 110′ andconfigured to interface with the third domain 123′ of the reagentcartridge 130′ described above, where the second thermal body 157′includes an array of recesses for surrounding storage volumes of thethird domain 123′ (e.g., to transfer heat for PCR and/or other heatingoperations for processes conducted within storage volumes of the thirddomain 123′). As shown in FIG. 2B and 6A, the set of thermal bodies canalso include a third thermal body 158′ (e.g., thermal plate) passingthrough an opening of the deck 110′ and configured to interface with theset of microwells 34 of the sample processing chip 132 described above,where the third thermal body 158′ provides a substantially planarsubstrate for uniform heat transfer to and from microwells of the sampleprocessing cartridge 130.

In variations, the set of thermal bodies can be coupled to heat sinkelements 155 (e.g., at sides of the thermal bodies away from interfaceswith reagent cartridges/sample processing cartridges), in order toprovide greater surface area for heat transfer. Furthermore, as shown inFIGS. 2D-2F, areas between the deck 110 and the base 180 described inmore detail below can include one or more fans 154 and/or ducts 153, inorder to provide thermal mechanisms for convective heat transfer awayfrom the set of thermal bodies as needed. Furthermore, in variationsdescribed above, one or more portions of the heating and coolingsubsystem 150 (e.g., thermal bodies, etc.) can include features thatfacilitate retention of corresponding cartridges (e.g., reagentcartridges, sample processing cartridges, etc.) in position.

In variations, one or more of the thermal bodies and/or other portionsof the heating and cooling subsystem 150 can be coupled to actuatorsthat move the thermal bodies into and out of thermal communication withelements supported by the deck 110; however, variations of the system100 can omit actuators of the heating and cooling subsystem 150.

2.1.6 Deck-Supported Element: Pumping Subsystem

As shown in FIGS. 1A, 2B, and 6A, the system 100 can include a pumpingsubsystem 157 (e.g., coupled to the deck 110 and/or base 180), whichfunctions to provide positive pressure and/or negative pressure todesired portions of the sample processing cartridge 130 described above.In more detail, the pumping subsystem 157 can function to drive fluidflow from the inlet reservoir 133 and into the sample processing chip132 of the sample processing cartridge 130. Additionally oralternatively, the pumping subsystem 157 can function to remove fluidfrom the waste containment region 137 of the sample processing cartridge130 and into an external waste receptacle. In variations, the pumpingsubsystem 157 can include one or more ports 58 (e.g., vacuum ports)configured to interface with the sample processing cartridge 130 throughopenings in the deck 110, one or more pumps (e.g., vacuum pumps,peristaltic pumps, etc.) coupled to the ports 58, one or more manifoldsto provide pressure driving pathways coupled to the pump(s), one or morepressure sensors configured to detect pressure levels along pressurepathways, and/or one or more control circuit elements configured tocontrol operation of the pumping subsystem 157 (e.g., with electricalcoupling to processing elements of the base 180 described in more detailbelow). As such, in variations, portions of the pumping subsystem 157not directly coupled to the sample processing cartridge 130. can besituated between the deck 157 and the base.

In variations, the port(s) 58 of the pumping subsystem 157 can becoupled (e.g., physically connected, fluidically connected, etc.)suitable regions of the sample processing cartridge 130 (e.g. inlet,wells, etc.), and can additionally or alternatively be coupled to thereagent cartridge 120, another fluidic pathway of the system 100, or anyother suitable component of the system 100.

In a first specific example, as shown in FIGS. 2F and 6A, the pumpingsubsystem 157 includes a pump 59 (e.g., peristaltic pump) arrangedbetween the deck 110 and the base 180, and coupled to the wastecontainment region at the waste outlet 51, through a coupling arrangedat a bottom broad surface of the base substrate 131 of the sampleprocessing cartridge 130, wherein the pump 59 can draw fluid through thesample processing chip 132 in forward and reverse directions by applyingnegative and/or positive pressure accordingly. Additionally oralternatively, the pumping subsystem 157 can draw fluid from the inletreservoir 133 and through the sample processing chip 132 at apredetermined pressure (e.g., −0.25 psi or −1 psi or −2.5 psi) accordingto triggering events associated with the fluid level detection subsystem159 described in more detail below.

In relation to the sample processing cartridge 130 and forces applied bythe lid-opening tool 145 to transition the lid 135 between closed andopen states, retention elements described in Section 2.1.4 above canprovide a retention force that balances/counteracts a force applied bythe lid-opening tool 145 to open the lid 135, as described above.Additionally or alternatively, one or more portions of the pumpingsubsystem 157 can retain the sample processing cartridge 130 in positionand provide a retention force that balances/counteracts a force appliedby the lid-opening tool 145 to open the lid 135, as described above. Ina specific example, the port 58 can couple with the sample processingcartridge 130 to provide a counteracting force to the lid-opening tool145. However, other portions of the system 110 can additionally oralternatively provide a counteracting force.

2.1.7 Deck-Supported Element: Fluid Level Detection Subsystem

As shown in FIGS. 1A, 2B, and 9 , the system 100 can include a fluidlevel detection subsystem 159 at least partially supported by the deck110 and configured to interface with the sample processing cartridge130. The fluid level detection subsystem 159 functions to detect and/ormeasure a fluid parameter (e.g. a binary presence of fluid, a volume offluid, a fluid flow rate, a fluid type etc.) associated with fluid atthe sample processing cartridge 130 and/or other fluid processingelements of the system 100. In variations, the fluid level detectionsubsystem 159 can include a fluid level sensor 63 coupled to fluid levelcontrol circuitry (e.g., with electrical coupling to processing elementsof the base 180 described in more detail below).

A unit of the fluid level sensor 63 can determine a fluid parameterassociated with the inlet reservoir 133 (e.g. fluid passing from theinlet reservoir to microwells) of the sample processing cartridge 130. Aunit of the fluid level sensor 63 can additionally or alternativelydetermine a fluid parameter associated with an outlet (e.g. fluidpassing from microwells to outlet, fluid passing from outlet to wastecontainment region, etc.) of the sample processing cartridge 130. A unitof the fluid level sensor 63 can additionally or alternatively determinea fluid parameter associated with the waste containment region (e.g.volume of fluid in waste containment region) of the sample processingcartridge 130, or any other suitable fluid parameter. The fluid levelsensor 63 can include any or all of: an optical sensor, pressure sensor,temperature sensor (e.g. to detect a fluid of certain temperature in afluidic pathway), or any other suitable sensor configured to detect thepresence of fluid and optionally determine a value associated with thefluid (e.g. volumetric flow rate, etc.) in the sample processingcartridge.

In a first variation, the system includes an optical sensor 63configured to detect the presence of fluid being transferred from theinlet to the set of wells. In an example, an infrared (IR)emitter/detector pair is used to determine the presence of fluid and avolume of the fluid being transferred (e.g. further based on theduration of time that the fluid is present) at the inlet reservoir 133of the sample processing cartridge 130.

In the specific example shown in FIGS. 2B and 9 , the fluid leveldetection subsystem 159 includes an IR emitter 63 a displaced from andopposing an IR detector 63 b across the inlet reservoir 133 of thesample processing cartridge 130, where the IR emitter 63 a and IRdetector 63 b are configured to be positioned within an undersideinternal portion of the base substrate 131 of the sample processingcartridge 130, to pass IR radiation across walls and an internal volumeof the inlet reservoir 133, thereby enabling fluid level detection.Variations of the fluid level sensor 63 can, however, be configured inanother suitable manner.

2.1.8 Deck-Supported Element: Separation Subsystem and Operation Modes

As shown in FIGS. 1A, 2B, and 10A-10C, the system 100 can include aseparation subsystem 160, which functions to facilitate separation oftarget material from non-target material (e.g., using magnetic forces,using other forces). In variations, the separation subsystem 160 caninclude embodiments, variations, and examples of components described inU.S. Application 62/866,726, titled “System and Method for TargetMaterial Retrieval from Microwells” and filed on 26 Jun. 2019, which isherein incorporated in its entirety by this reference. However,variations of the separation subsystem 160 can additionally oralternatively include other components.

In one variation, as shown in FIGS. 10A-10C, the separation subsystem160 can include a first body 161 including an interface 162 to the fluidhandling subsystem (e.g., pipette interface) of the gantry 170 describedbelow, and a magnetic distal region 163 configured to provide magneticforces for target material separation. In this variation, the magneticdistal region 163 can be configured to couple with one or more units ofthe magnetic sleeves 1410 (e.g., of tool container 140) described above,where the magnetic sleeves 1410 can be disposable elements. Furthermore,the interface 162 can be configured to couple to a pipetting headcoupled to the gantry 170 described in more detail below, in order tofacilitate target or non-target material retrieval by way of magneticforces, fluid aspiration, and/or fluid delivery operations provided bythe pipetting head. As such, the system 100 can include a separationmode in which the gantry 170 transports the first body 161, coupled tothe magnetic sleeve 1410, between the sample processing cartridge 130and the reagent cartridge 120 for magnetic separation of target materialfrom a sample. Furthermore, embodiments of methods implemented using theseparation subsystem 160 can produce rapid retrieval of target material,with a retrieval efficiency of >90% where only magnetic particlescoupled to target material (or non-target material) of the sample areretrieved. The separation subsystem 160 can thus function to produceincreased selective retrieval efficiency can thus reduce downstreamcosts in relation to processing reagent and other material costs (due toreduced volumes needed, due to reduced splits in biochemistry reactions)and processing burden.

As shown in FIG. 10A, the first body 161 can include an interface 162 tothe fluid handling subsystem of the gantry 170 described below, wherethe interface includes a coupling region that complements acorresponding coupling region of the fluid handling subsystem. Thecoupling region of the interface 162 can operate by: a magnetic couplingmechanism; a press fit; a snap fit, a screwing mechanism; a male-femaleconnection; or another suitable mechanism for providing reversiblecoupling with the fluid handling subsystem.

The magnetic distal region 163 of the first body 161 can include or becomposed of a material for providing a permanent magnet, or canalternatively be configured as an electromagnet (e.g., with coupling tosuitable electronics of the system Dm). In variations, the magneticdistal region 163 can be composed of one or more of: alnico, neodymium,neodymium iron boron, samarium cobalt, ferrite, and any other suitablemagnetic material. In morphology, the magnetic distal portion 163 cancomplement a morphology of the magnetic sleeve 1410, such that units ofthe magnetic sleeve 1410 can couple (e.g., reversibly couple) with themagnetic distal portion. Furthermore, the morphology and poleconfiguration of the magnetic distal portion 163 is such that nearlynormal magnetic force is applied to majority of the target microwellsfrom where entrapped particles are being removed.

The magnetic sleeve 1410 can include a first region 1410 a configured tointerface with the sample processing chip 132, for instance, throughaccess region 134, in order to enable transfer of material from thesample processing chip 132. The magnetic sleeve 1410 can also include asecond region 1410 b for coupling with the magnetic distal portion 163of the first body 161, and an internal cavity 1410 c passing from thefirst region to the second region. The magnetic sleeve 1410 functions toprovide structures that separate the first body 163 from physicallycontacting wells or other sensitive material of the sample processingchip 132, and to support application of a magnetic field to the desiredregions for retrieval of target material (or non-target material). Themagnetic sleeve 1410 can also function to prevent sample crosscontamination, by serving as a disposable component that can bediscarded between uses of the system 100.

The magnetic sleeve 1410 can be morphologically prismatic with aninternal cavity 1410 c, where the cross section of the magnetic sleeve1410 along its longitudinal axis is defined by a polygonal perimeter, anellipsoidal perimeter, an amorphous perimeter, or a boundary of anyother suitable shape (e.g., closed shape, open shape). The cross sectionof the magnetic sleeve 1410 can complement a shape of a footprint of themicrowell region of the sample processing chip 132, but mayalternatively not complement a shape corresponding to the sampleprocessing chip 132. The magnetic sleeve 1410 preferably has a wallthickness that supports application of a magnetic force, from themagnetic distal portion 163, to the sample processing chip 132interfacing with the first region 1410 a of the magnetic sleeve 1410.The wall thickness can be constant or non-constant along the length ofthe adaptor 210. In examples, the wall thickness can range from 0.2 to 3mm thick; however, in other examples, the wall thickness can have anyother suitable thickness. The surface of the magnetic sleeve 1410 thatreceives the magnetic particles is made smooth (say surface finishbetter than SPIB1) such that the small magnetic particles (1-3 micron)do not gets entrapped in the surface during the bead capture onto itssurface and subsequent release to another receptacle.

The magnetic sleeve 1410 can additionally or alternatively includestructural features that enable separation operation modes of theseparation subsystem 160. For instance, in relation to release of themagnetic sleeve 1410 from the pipetting head (described in more detailbelow), the magnetic sleeve 1410 can include a protrusion 1410 dconfigured to allow another object (e.g., sleeve stripping tool 165) toprovide a force against the protrusion 1410 d to release the magneticsleeve 1410 from the first body 161.

As described above, the magnetic sleeve 1410 couples, at a first region1410 a, to a region of the sample processing chip 132 exposed throughaccess region 134, in order to facilitate application of magnetic forceto the region, and to enable drawing of material (e.g., target ornon-target material coupled to magnetic particles) into the magneticsleeve 1410 for further downstream processing. The magnetic sleeve 1410can thus include a seal at the first region 1410 a, in order tofacilitate mechanisms for drawing target material from the sampleprocessing chip 132 into the magnetic sleeve 1410. The seal can be aseparate element or an element integrated with the magnetic sleeve 1410.The magnetic sleeve 1410 can, however, omit a seal at the first region1410 a.

The magnetic sleeve 1410 can be composed of a polymeric material (e.g.,plastic) that does not adversely affect the magnetic field applied bythe magnetic distal portion 163 during operation. The magnetic sleeve1410 can additionally or alternatively include (e.g., include particlesof) or be composed of a material (e.g., metallic material) that ismagnetic or can produce an induced magnetic field to supportapplications of use of the system 100. The magnetic sleeve 1410 canadditionally or alternatively be composed of any other suitablematerial. Distributions of the material(s) of the magnetic sleeve 1410can be homogenous or non-homogenous through the body of the adaptor, inrelation to desired magnetic effects at the capture region of the sampleprocessing chip 132. The internal cavity iztioc of the magnetic sleeve1410 can include a medium (e.g., magnetic medium, etc.), or canalternatively not include any medium.

As shown in FIGS. 1A, 2B, and 11A-11B, in variations, the separationsubsystem 160 can include a magnet subsystem 166 including a set ofmagnets 167 within a housing 168, where the magnet subsystem 166 furtherincludes a magnet actuator 169 configured to move the set of magnets 167relative to the deck 110 (e.g., through in opening in the deck no), andinto/out of alignment with one or more separation reservoirs 129 of thereagent cartridge 120 described above. The magnet actuator 169 can alsobe coupled to control circuitry (e.g., at the base 180) described inmore detail below. Furthermore, the magnet actuator 169 can beconfigured to transition the set of magnets between a retracted stateand an extended state, wherein in the extended state, the set of magnetspasses into the first region of the deck (e.g., as shown in FIGS. 11Aand 11B). As such, the separation subsystem 160 can also includeelements that are supported by the deck 110 and/or base 180, in order toenable operations for separating target material from non-targetmaterial.

In variations, the set of magnets 167 can include one or more permanentmagnets and/or electromagnets (e.g., with coupling to suitableelectronics of the system loo). Permanent magnets can be composed of oneor more of: alnico, neodymium, neodymium iron boron, samarium cobalt,ferrite, and any other suitable magnetic material.

In the example shown in FIGS. 11A-11B, the set of magnets 167 caninclude a first subset of magnets 167 a arranged in a linear array(e.g., for performance of purification operations at the reagentcartridge 120), where the positions of the first subset of magnets 167 acorrespond to positions of volumes of the fifth domain 125′ for particleseparation/purification, described in relation to the reagent cartridge120′ above and workflows in Section 3 below. In the example shown inFIG. 11A-11B, the set of magnets 167 also includes a second subset ofmagnets 167 b (e.g., one or more magnets) displaced from or otherwiseoffset from an axis associated with the first subset of magnets 167 a,in order to interact with a separation reservoir 129 a of the reagentcartridge 120 (e.g., for initial bead retrieval). The set of magnets 167can, however, be arranged in another suitable manner (e.g., in relationto distributed arrays, in relation to number, etc.) in relation toproviding suitable interactions with separation reservoirs 129 of thereagent cartridge 120 or other cartridges.

The housing 168 functions to surround the set of magnets 167, and toprovide smooth operation in relation to transitioning the set of magnets167 into/out of alignment with corresponding portions of the reagentcartridge 120. Thus, as shown in FIG. 11B, in relation to configurationswhere there is a first subset of magnets 167 a and a second subset ofmagnets 167 b, the housing 168 can include a first surface (e.g., firstplanar surface) tracking the first subset of magnets 167 a, and a secondsurface (e.g., second planar surface) tracking the second subset ofmagnets 167 b, wherein the first surface 168 a and the second surface168 b are angled away from each other. In this variation, a pair ofopposing walls can extend from the first surface and the second surface,in order to promote smooth operation (e.g., sliding operations) of thehousing 168 and magnets through the deck 110 in order to interface withthe reagent cartridge 120.

In relation to the reagent cartridge 120′, as shown in FIG. 3A and 11B,volumes of the reagent cartridge 120 configured for magnetic separationcan each include a planar surface 128 a, or other surface complementaryto the housing 168 at sides configured to be closest to the housing 168during operation (e.g., in the extended magnet states). Furthermore,volumes of the reagent cartridge 120 configured for magnetic separationcan each include a second surface 128 b (e.g., curved surfaces)displaced away from the housing 168 for aspiration and/or delivery offluids by a pipettor coupled to the gantry 170 described below. Crosssections taken longitudinally through separation volumes/reservoirs 129of the reagent cartridge 120 can further be tapered toward a base of thereagent cartridge 120, such that separation operations require a lowervolume of fluid and/or provide more efficient aspiration and separationof target from non-target material.

As shown in FIGS. 12A through 12J, the separation subsystem 160 canprovide a sequence of operation modes for material separation, where, asshown in FIG. 12A, the operation modes involved specific systemstructure configurations of: a first body 161 coupled with a pipettinghead or other actuatable component (e.g., described in relation to thegantry 170 below), the first body having a magnetic distal region 163, amagnetic sleeve 1410, a sleeve stripping tool 165, a separationreservoir 129, and a magnet 167 b of the set of magnets 167 describedabove.

In more detail, as shown in FIG. 12B, the separation subsystem 160 canprovide a first operation mode 164 a, where the first operation mode 164a is a baseline operation mode in which the first body 161 is uncoupledfrom a pipette interface or other actuatable component (e.g., describedin relation to the gantry 170 below) and the magnetic distal region 163of the first body 161 is uncoupled from the magnetic sleeve 1410. Themagnetic sleeve 1410 is further retained by sleeve stripping tool 165above the separation reservoir 129 (or in variations, at anotherposition), and the magnet 167 b is displaced away from the separationreservoir 129 (e.g., by magnet actuator 169 described above).

As shown in FIG. 12C, the separation subsystem 160 can provide a secondoperation mode 164 b, where the second operation mode 164 b is aninitializing operation mode in which the first body 161 is coupled witha pipette interface or other actuator interface 6 (e.g., described inrelation to the gantry 170 below) and the magnetic distal region 163 ofthe first body 161 is uncoupled from the magnetic sleeve 1410. Themagnetic sleeve 1410 is further retained by sleeve stripping tool 165above the separation reservoir 129, and the magnet 167 b is displacedaway from the separation reservoir 129 (e.g., by magnet actuator 169described above).

As shown in FIG. 12D, the separation subsystem 160 can provide a thirdoperation mode 164 c, wherein, in the third operation mode 164 c, thefirst body 161 is coupled with a pipette interface or other actuatorinterface 6 (e.g., described in relation to the gantry 170 below) andmoved into alignment with the separation reservoir 129. In the thirdoperation mode 164 c, the magnetic distal region 163 of the first body161 is coupled with the magnetic sleeve 1410 above the separationreservoir 129 in the retained position of the magnetic sleeve 1410. Inthe third operation mode 164 c, magnet 167 b is displaced away from theseparation reservoir 129 (e.g., by magnet actuator 169 described above).

As shown in FIG. 12E, the separation subsystem 160 can provide a fourthoperation mode 164 d, wherein, in the fourth operation mode 164 d, thefirst body 161 is coupled with a pipette interface or actuator interface6 (e.g., described in relation to the gantry 170 below) and the magneticdistal region 163 of the first body 161 is coupled with the magneticsleeve 1410 above the separation reservoir 129. In the fourth operationmode 164 d, the pipetting head (or other actuatable component) moves thefirst body 161 and magnetic sleeve 1410 out of the retained positionprovided by the sleeve stripping tool 165, to prepare for extraction ofmaterial (e.g., functionalized particles from the sample processingcartridge) and/or delivery of material from the pipette interface,coupled to the first body and magnetic sleeve, into the separationreservoir 129. In the fourth operation mode 164 d, magnet 167 b isdisplaced away from the separation reservoir 129 (e.g., by magnetactuator 169 described above).

As shown in FIG. 12F, the separation subsystem 160 can provide a fifthoperation mode 164e, wherein, in the fifth operation mode 164 e, thefirst body 161 is coupled with a pipette interface or actuator interface6 (e.g., described in relation to the gantry 170 below) and the magneticdistal region 163 of the first body 161 is coupled with the magneticsleeve 1410 above the separation reservoir 129. In the fifth operationmode 164 d, the pipette interface (or other actuatable component)delivers fluid from the pipetting head into the separation reservoir129, and the magnetic sleeve 1410, still coupled with the first body 161(and coupled to functionalized particles), is submerged within the fluidin the separation reservoir 129. In the fifth operation mode 164e,magnet 167 b is displaced away from the separation reservoir 129 (e.g.,by magnet actuator 169 described above).

As shown in FIG. 12G, the separation subsystem 160 can provide a sixthoperation mode 164f, wherein, in the sixth operation mode 164 f, thefirst body 161 is coupled with a pipette interface or actuator interface6 (e.g., described in relation to the gantry 170 below) and the magneticdistal region 163 of the first body 161 is coupled with the magneticsleeve 1410 above the separation reservoir 129. In the sixth operationmode 164 f, the pipette interface (or other actuatable component) movesthe magnetic sleeve 1410, still coupled with the first body 161 backinto a retained position at the sleeve stripping tool 165 , and themagnetic sleeve 1410 (still coupled with functionalized particles) issubmerged within the fluid in the separation reservoir 129. In the sixthoperation mode 164 f, magnet 167 b is displaced away from the separationreservoir 129 (e.g., by magnet actuator 169 described above).

As shown in FIG. 12H, the separation subsystem 160 can provide a seventhoperation mode 164 g, wherein, in the seventh operation mode 164 g, thefirst body 161 is coupled with a pipette interface or actuator interface6 (e.g., described in relation to the gantry 170 below) and the magneticdistal region 163 of the first body 161 is coupled with the magneticsleeve 1410 above the separation reservoir 129. In the seventh operationmode 164 g, the magnetic sleeve 1410, still coupled with the first body161 is in a retained position at the sleeve stripping tool 165, and themagnetic sleeve 1410 (with functionalized particles) is submerged withinthe fluid in the separation reservoir 129. In the seventh operation mode164 g, magnet 167 b is displaced toward the separation reservoir 129(e.g., by magnet actuator 169 described above) for to prepare forattraction and retention of target or non-target material coupled to thefunctionalized particles of the fluid against a wall 128 a of theseparation reservoir 129.

As shown in FIG. 121 , the separation subsystem 160 can provide aneighth operation mode 164 h, wherein, in the eighth operation mode 164h, the first body 161 is coupled with a pipette interface or actuatorinterface 6 (e.g., described in relation to the gantry 170 below), andis being moved away from the separation reservoir 129 to be removed andreplaced with a suitable tip from the tool container described above. Inthe eighth operation mode 164 h, the magnetic distal region 163 of thefirst body 161 is uncoupled from the magnetic sleeve 1410 above theseparation reservoir 129, by having the pipetting head move the firstbody 161 away from the magnetic sleeve 1410 while the magnetic sleeve1410 is retained in position at the sleeve stripping tool 165. In theeighth operation mode 164 h, the magnetic sleeve 1410 is submergedwithin the fluid in the separation reservoir 129. In the eighthoperation mode 164 h, magnet 167 b is still positioned in proximity tothe separation reservoir 129 (e.g., by magnet actuator 169 describedabove) for retention of target or non-target material coupled tofunctionalized particles of the fluid against a wall 128 a of theseparation reservoir 129. In the eighth operation mode 164 h, the magnet167 b draws target material toward the bottom of the separationreservoir 129 (e.g., for later extraction from the bottom by thepipettor, for retention while the pipettor draws material unbound by themagnet 167 b).

As shown in FIG. 12J, the separation subsystem 160 can provide a ninthoperation mode 164 i, wherein, in the ninth operation mode 164 i, thepipetting head/actuator interface 6 is coupled with a suitable tip andmoved into the separation reservoir 129 to aspirate material from theseparation reservoir 129. In the ninth operation mode 164 i, themagnetic sleeve 1410 is still retained in position above the separationreservoir 129 at the sleeve stripping tool 165 and submerged within thefluid in the separation reservoir 129. In the ninth operation mode 164i, magnet 167 b is moved away from the separation reservoir 129 (e.g.,by magnet actuator 169 described above) in coordination with aspirationof material by the pipetting head.

FIGS. 13A through 13D depict additional views of configurations of amagnetic sleeve 1410 with respect to sleeve stripping tool 165 of aseparation reservoir 129 of a reagent cartridge 120, in relation tooperation modes described above.

Variations of the separation subsystem 160 can, however, includeelements and provide modes of operation for target material retrievalbased upon one or more of: gravitational forces, buoyant forces,centrifugal forces, chemical separation, and/or any other suitableseparation approaches. In yet another embodiment, target materialretrieval operation by the separation subsystem 160 may be used totransfer target particles from the microwell chip to another substrateor another new empty microwell chip while keeping the relative spatiallocations of the different particles being transferred.

2.2 System—Gantry

As shown in FIGS. 1A-1B, 2A, and 2C-2F, the system 100 can include agantry 170 coupled to the deck no, which functions to support and/orenable actuation of one or more tools for various interactions withelements of the deck no, along a set of axes. In variations, the gantry170 provides one or more rails/tracks for moving tools, such as pipettor174 with pipette interface described below, in three dimensional space(e.g., a three dimensional volume bound by the first side of the deck).In variations, tools actuated using the gantry 170 can be moved relativeto the sample processing cartridge 130, reagent cartridge 120, toolcontainer 140, or other elements, for transfer of materials (e.g. cells,reagents, particles, etc.) across different components supported by thedeck no. Additionally or alternatively, tools supported by the gantry170 can be used for reading of barcodes associated with variousdisposables supported by the deck no (e.g., in relation to identifyingproper setup of a run, in relation to inventory management, etc.). Thegantry 170 preferably enables movement of one or more tools along one ormore axes (e.g., X and Y axes shown in FIG. 2A) parallel to broadsurfaces of the reagent cartridge 120, sample processing cartridge 130,and tool container 140, and additionally along an axis (e.g., Z axisshown in FIG. 2A) perpendicular to the broad surfaces. The gantry 170can additionally or alternatively enable movement along a subset ofthese directions or along any other suitable direction. In order toenable movement, the gantry 170 includes or is otherwise coupled to oneor more motors (e.g., motors for each axis or direction of movement),one or more encoders for position identification in each axis ordirection of movement, and/or one or more switches (e.g., opticalswitches for each axis) for control of the gantry 170 (e.g., where theswitches are electrically coupled with control circuity described inrelation to the base 180 below).

In a first variation (e.g., as shown in FIGS. 2A-2B and 2D-2F), thegantry 170 includes a three dimensional track assembly (X-Y-Z trackassembly) including an X-rail 171 for movement of tools along an X-axis,a Y rail 172 for movement of tools along a Y-axis, and a Z-rail 173 formovement of tools along a Z-axis. In the variation shown in FIG. 2A, theX-rail 171 is coupled with and slides along the Y-rail 172, and theZ-rail 173 is coupled with and slides along the X-rail 171; however, inother variations, the X-rail 171, Y-rail 172, and Z-rail 173 can becoupled and interact with each other in another suitable manner. In thevariation shown in FIG. 2A, the gantry 170 is configured to move tools,using the X-rail 171 and the Y-rail 172, into alignment with elements ofthe deck no, and is configured to move tools, using the Z-rail 173 foraccessing elements of the deck (e.g., portions of the reagent cartridge120, elements of the sample processing cartridge 130, tools of the toolcontainer 140, etc.). In some variations, the gantry 170 can beconfigured to return to a parked position (e.g., once a protocol iscompleted, when the system is turned off, before the lid is opened,etc.), where the parked position allows the user to access appropriateareas of the deck (e.g., for refilling tips, filling tubes withreagents, removing a microwell cartridge, etc.). However, the gantry 170can have another suitable baseline position. Various operation modes ofthe gantry 170 are further described in relation to workflows of Section3 below.

2.2.1 Gantry—Pipettor

As shown in FIG. 2A, the gantry 170 can include and/or or be configuredto interact with a pipettor 174, which functions to hold, move, and/orotherwise interact with any number of tips or other tools, such as thoseof the tool container 140 described above. In variations, the pipettor174 assembly can include one or more of: a pump (e.g., displacementpump) for providing pressure differentials for delivery and aspirationof fluids, a pressure sensor for sensing pipetting pressure, a levelsensor for sensing fluid level within the pipettor 174, a tip detector(e.g., to enable determination of presence or absence of a tip coupledto the pipettor 174), and a tip ejection motor coupled to a tip ejectorfor removing tips from the pipettor 174. As shown in FIG. 2A, thepipettor 174 can be coupled to the Z-rail 173 of the gantry 170;however, in other variations, the pipettor 174 can additionally oralternatively be coupled to other portions of the gantry 170.

The pipettor 174 is preferably operable in an automated fashion (e.g.,motorized, mechanized, self-operating, etc.) and can be configured tocontrol any or all of the following predetermined parameters: volume(e.g. dispensing exact volumes, aspirating exact volumes), a heightabove the well at which each material is dispensed (e.g. priming bufferis dispensed between 0.25 and 0.3 millimeters above the top of eachwell, cell suspension is dispensed at a height of 0.25 millimeters abovethe top of each well, etc.), or can control any other suitable propertyaccording to any suitable parameter. Additionally or alternatively, thepipettor 174 can be configured to operate in a manual fashion (e.g.,according to a user, with user intervention, held and used by a user,etc.) or in any suitable way. In yet another embodiment, the pipettor174 may be used to pick up one or more tools associated with the toolcontainer, such as any or all of: a mechanical tool, magnetic tool, anoptical tool, and any other suitable tool. The tools can be moved by thepipettor 174 to the reagent cartridge and/or the microwell cartridgesuch that the tool(s) can perform specific mechanical/magnetic and/oroptical functions with respect to specific contents of the reagentcartridge or microwell cartridge.

2.2.2 Gantry—Other Tools

Additionally, the gantry 170 can include or support one or more toolsdescribed in relation to other elements above. For instance, the gantry170 can be coupled with a unit of lid-opening tool 145 (shown in FIG.2B) for transitioning the lid 135 of the sample processing cartridge 130between open and closed states. The gantry 170 can additionally oralternatively be coupled with a magnetic head actuator 175 (shown inFIG. 2B) associated with the separation subsystem 160 described above(e.g., in relation to actuation of the first body 161 with magneticdistal region 163) for separation of target material from non-targetmaterial.

In some variations, the gantry 170 can directly or indirectly be coupledwith a camera 176 (e.g., camera coupled with a light), which functionsto enable reading of tags (e.g., barcodes) associated with variousdisposables supported by the deck 110 (e.g., in relation to identifyingproper setup of a run, in relation to inventory management, etc.).Additionally or alternatively, the camera 176 can include functionalityfor transmitting image data capturing configurations of elements at thedeck no without reading of specific tags. As shown in FIGS. 2A and 2F,the camera 176 can be coupled to the Z-rail 173 with pipettor 174described above, such that the camera 176 has a field of view associatedwith objects that the pipettor 174 is aligned with. However, motion ofthe camera 176 can alternatively be uncoupled from the pipettor 174,such that the camera 176 can be moved independently of the pipettor 174.In variations, the camera 176 can include components described inapplications incorporated by reference above. The camera image can alsobe used as an additional feedback mechanism for the precise movement ofthe gantry 170 and/or its components (e.g., pipettor, tools, etc.) toreach a desired location, thereby dramatically improving the accuracyand precision of the motion system (e.g., to less than 100 microns, lessthan 50 microns, less than 25 microns, less than 10 microns, less than 5microns, less than 1 micron, etc.).

Additionally or alternatively, as shown in FIGS. 1A and 1B, the gantry170 can include various sensors for monitoring environmental or otherparameters of the system 100. For instance, in variations, the sensorscan include one or more of: temperature sensors, humidity sensors,pressure sensors, optical sensors, vibration sensors, and other suitablesensors. The sensors can, however, be positioned relative to the system100 in another suitable manner (e.g., uncoupled from the gantry 170,positioned at the deck 110, positioned at the base 180, etc.).

2.3 System—Base

As shown in FIG. IA, the system 100 can include a base 180, whichfunctions to support control and processing architecture associated withelements of the deck 110 and gantry 170 described above. In variations,the base 180 can support control and processing architecture for one ormore system functions including: pressure modification for fluiddelivery throughout the sample processing cartridge 130 and/or pipettor174; fluid level sensing (e.g., at the sample processing cartridge 130,at the pipettor 174, at various storage volumes of the reagent cartridge120, etc.); actuation of lid opening mechanisms of the sample processingcartridge 130; thermocycling and/or other heating functions for thereagent cartridge 120 and/or sample processing cartridge 130; coolingfunctions for the reagent cartridge 120 and/or sample processingcartridge 130; separation functions (e.g., elution, magnetic separation,other separation, etc.); functions for control of the gantry 170;functions involving receiving sensor signals and returning outputs;functions involving receiving sensor signals and executing variousactions; functions for transitioning system doors between various states(e.g., open states, closed states, locked states, unlocked states,etc.); functions associated with system power management; functionsassociated with system status indication elements (e.g., lights, audiooutput devices, visual output devices, etc.); functions associated withsystem input devices (e.g., buttons, keyboards, keypads, mice,joysticks, switches, touch screens, etc.); functions associated withdisplay devices; functions associated with system data storage devices;functions associated with system transmission devices (e.g., wiredtransmission devices, wireless transmission devices, etc.); and othersuitable functions.

In variations, the base 180 can thus support an electronics subsystem(e.g. PCB, power source, communication module, encoder, etc.) associatedwith a processing architecture (e.g. onboardthe system, separate fromthe system, etc.), or any other suitable component, where the processingarchitecture can include any or all of: processors (e.g.microprocessors), controllers (e.g. microcontrollers), memory, storage,software, firmware, or any other suitable component. Additionally, theprocessing subsystem can include a machine vision module, whichfunctions to read tags, verify protocols, perform error detection (e.g.detect that reagents do not match an assigned protocol), or perform anyother function.

For instance, in an example operation flow, an operator can initiate theperformance of the protocol (e.g., by pushing a button of the system, byinteracting a touch-sensitive display of the system to make a selection,etc.). A barcode reader performs an error detection protocol by scanningtags of the deck elements (e.g, reagent cartridge, sample processingcartridge, tool container, etc.) and comparing with the protocolselected by the user; if the tags do not match the selected protocol, anotification can be transmitted to the user, and if the tags arecorrect, the protocol can begin. At this point, the operator may nolonger needed. According to one or more workflows, some of which aredescribed in Section 3 below, the correct types and volumes of materials(e.g., reagents/samples) are added to or removed from the sampleprocessing cartridge at the correct times in an automated fashion. Oncethe protocol is complete, the operator can proceed with collectingand/or processing the contents of the microwell cartridge as desired,and/or setting up a new run. Variations of methods and workflows enabledby the system 100 are further described below.

Embodiments, variations, and examples of control and processingarchitecture are further described in U.S. application Ser. No.16/048,104, filed 27 Jul. 2018; U.S. application Ser. No. 16/049,057,filed 30 Jul. 2018; U.S. application Ser. No. 15/720,194, filed 29 Sep.2017; U.S. application Ser. No. 15/430,833, filed 13 Feb. 2017; U.S.application Ser. No. 15/821,329, filed 22 Nov. 2017; U.S. applicationSer. No. 15/782,270, filed 12 Oct. 2017; U.S. application Ser. No.16/049,240, filed 30 Jul. 2018; U.S. application Ser. No. 15/815,532,filed 16 Nov. 2017; U.S. application Ser. No. 16/115,370, filed 28 Aug.2018, U.S. application Ser. No. 16/564,375, filed 9 Sep. 2019, and U.S.application Ser. No. 16/816,817, filed 12 Mar. 2020, as incorporated byreference above.

2.4 System—Functional Coupling Between Deck, Gantry, and Base

In variations, the deck 110, gantry 170, and base 180 of the system canbe functionally coupled to provide various functions. Functionalcoupling can be electromechanical, mechanical, electrical, magnetic,pneumatic, fluidic, optical, and/or of other coupling means, accordingto various modes of operation. FIGS. 14A-14C depict functional couplingfor a variation of a system, as described below.

FIG. 14A depicts an example of mechanical, electrical, pneumatic,optical, and magnetic communication between various elements of the deck110, gantry 170, and base 180. In more detail, as shown in FIG. 14A, thegantry 170 supports actuation-associated elements including a Z-axis arm801 associated with Z-rail 173 described above, 3-axis gantry motors802, 3-axis encoders 803, 3-axis optical switches 804, a magneticactuator 805, and a lid-opening tool 806. The gantry 170 furthersupports pipetting elements including: a pipettor 174, a displacementpump 808, a pipette tip detector 809, a pipette tip ejection motor 810,a level sensor 811, and a pressure sensor 812. The gantry 170 alsosupports a camera and light 176 and a set of sensors 814 includingtemperature and humidity sensors. As shown in FIG. 14A, the deck 110supports a sample processing cartridge 130, a reagent cartridge 120, anda tool container 140 including a set of tools 865. As shown in FIG. 14A,the base 180 supports a first computing subsystem 815, one or moreinterface boards 816, and gantry-control circuitry 817. Mechanical,electrical, pneumatic, optical, and magnetic communication betweenvarious elements of the deck 110, gantry 170, and base 180 are depictedwith various line types in FIG. 14A.

FIG. 14B depicts an example of mechanical, fluidic, magnetic, andpneumatic communication between various elements of the deck 110 andbase 180. In more detail, as shown in FIG. 14B, the deck 110 supports areagent cartridge 120 including: a storage volume for ambient reagentsand functional beads 818, a sample storage volume 819 for receiving araw sample 820, a storage volume for storing chilled reagents and/orresults of sample processing 821, a set of storage volumes forseparation particles and associated separation reservoirs 822, and a setof storage volumes for PCR 823, with associated PCR covers 824; a toolcontainer 140 including a set of tools (e.g., pipette tips) 825; asample processing cartridge 130; and a set of deck tools 826 including aheater head 827, a suction head 828, and a magnetic head 829. As shownin FIG. 14B, a set of elements interface the deck 110 with the base 180,where the set of elements can include one or more of: a vacuum port 830,a pinch actuator 831, a sample processing cartridge thermal body 832(configured to mate with the microwell region of the sample processingcartridge 130 or other region of the sample processing cartridge 130), afirst reagent cartridge thermal body 833 (configured to mate with adomain of the reagent cartridge 120, at the first region in, for chilledreagents), a second reagent cartridge thermal body 834 (configured tomate with a domain of the reagent cartridge 120, at the first region in,for PCR thermocycling), a fluid level sensor 836, and a magnet 835 forseparation processes. Mechanical, fluidic, magnetic, and pneumaticcommunication between various elements of the deck 110 and base 180 aredepicted with various line types in FIG. 14B.

FIG. 14C depicts an example of electrical communication between variouselements of the deck 110 and base 180. In more detail, as shown in FIG.14C, the deck 110 supports a sample processing cartridge 130; a reagentcartridge 120 including: a storage volume for storing chilled reagentsand/or results of sample processing 821, a set of storage volumes forseparation particles and associated separation reservoirs 822, and a setof storage volumes for PCR 823 with associated PCR covers; and a set ofdeck tools 826 including a heater head 827. As shown in FIG. 14C, a setof elements interface the deck 110 with the base 180, where the set ofelements can include one or more of: a vacuum port 830, a pinch actuator831, a sample processing cartridge thermal body 832, a first reagentcartridge thermal body 833, a second reagent cartridge thermal body 834,a magnet 835 for separation processes, and a fluid level sensor 836.

In more detail, the vacuum port 830 is coupled to a pumping subsystem837 which includes a vent valve manifold 838, and a pressure sensor 839.The sample processing cartridge thermal body 832 is coupled to athermocycler 840 of a heating and cooling subsystem, which includes aheater 841 (e.g., Peltier heater, thermocouples, heat sinks, and fans)and thermal control circuitry 842. The first reagent cartridge thermalbody 833 is coupled to a cooling device 843 of a heating and coolingsubsystem, which includes a cooling element 844 (e.g., Peltier cooler,thermocouples, heat sinks, and fans) and thermal control circuitry 845.The second reagent cartridge thermal body 834 is coupled to a heatingdevice (e.g., for on-boardPCR) of a heating and cooling subsystem, whichincludes a first heater 846 (e.g., Peltier heater, thermocouples, heatsinks, and fans), a second heater 847 coupled to the heat sink, andthermal control circuitry 848. The magnet 835 is coupled to a magnetactuator 849. Finally, the fluid level sensor 836 is coupled to a fluidlevel controller 850.

As shown in FIG. 14C, the base 180 further supports a first computingsubsystem 815, one or more interface boards 816, a speaker 851, a doorlocking element 852 (interfacing with a door magnet 853 to provideoperation modes of a door 90, as described above), a power inlet 854coupled to a power supply 855 coupled to AC mains 856, a power switch857, indicator lights 858 (e.g., for power/ground effects), exhaust fans859, a set of ports (e.g., including an ethernet port 860 and USB ports861), and a display 862 (e.g., touchscreen display). Elements of thebase can further interface with off-system accessories (e.g., inputdevices 863, storage devices 864). Electrical, communication betweenvarious elements of the deck 110 and base 180 are depicted with variousline types in FIG. 14C.

3. Method

As shown in FIG. 15 , an embodiment of a method 300 for sampleprocessing includes: positioning a reagent cartridge, a tool container,and a sample processing cartridge at a deck of a sample processingsystem S310; transmitting content of the reagent cartridge between thesample processing cartridge by using tools of the tool container in anoperational sequence S320; and processing a sample at the sampleprocessing cartridge and/or reagent cartridge S330. Additionally oralternatively, the method 300 can include any or all of the processesdescribed in U.S. application Ser. No. 16/048,104, filed 27 Jul. 2018;U.S. application Ser. No. 16/049,057, filed 30 Jul. 2018; U.S.application Ser. No. 15/720,194, filed 29 Sep. 2017; U.S. applicationSer. No. 15/430,833, filed 13 Feb. 2017; U.S. application Ser. No.15/821,329, filed 22 Nov. 2017; U.S. application Ser. No. 15/782,270,filed 12 Oct. 2017; U.S. application Ser. No. 16/049,240, filed 30 Jul.2018; U.S. application Ser. No. 15/815,532, filed 16 Nov. 2017; U.S.application Ser. No. 16/115,370, filed 28 Aug. 2018, U.S. applicationSer. No. 16/564,375, filed 9 Sep. 2019, and U.S. application Ser. No.16/816,817, filed 12 Mar. 2020, which are each incorporated in theirentirety by this reference.

The method is preferably performed with an embodiment, variation, orexample of the systems described above (e.g., in relation totransmission of content between various elements and/or sampleprocessing), but can additionally or alternatively be performed with anyother suitable system. The method is further preferably at leastpartially automated (e.g., requires user to load reagents and select aprotocol, requires no user intervention, etc.), but one or more portionscan additionally or alternatively be manually performed (e.g., forquality control steps, for all protocols, for rare protocols, etc.).

Specific workflows associated with the method 3 0o and system elementsdescribed above are described in further detail below, where samples(e.g., samples including cell-derived material, proteins, mRNAs,proteins and mRNA; samples that include multiple samples each taggedwith multiplexing barcodes; samples that include encapsulated particlesfrom either cell or non-cell derived biomarkers, etc.) can be processedaccording to the workflows (e.g., workflows in Sections 3.1-3.3 below),followed by library preparation workflows (e.g., workflow in Section 3.4below), followed by next generation sequencing (NGS).

3.1 Method—Example Workflow for a 3′ Protocol for mRNA Synthesis/cDNAAmplification

As shown in FIG. 16 , a variation of the method, configured for mRNAsynthesis/cDNA amplification 300′ can include: performing a runpreparation operation, wherein the run preparation operation configuresa system for performing an mRNA synthesis/cDNA amplification protocolS305′; priming a sample processing cartridge of the system uponcompletion of the run preparation operation S310′; co-capturing a set ofcells of a sample with a set of functionalized particles, in single cellformat, at the sample processing cartridge S315′; lysing the set ofcells followed by binding released mRNA from the lysed cells with thefunctionalized particles (e.g., barcoded microspheres) (e.g., withassociated washing the functionalized particles of any unbound mRNA)S320′; performing a reverse transcription operation; retrieving the setof functionalized particles, with associated bound target content, fromthe sample processing cartridge S325′; performing an exonucleasetreatment operation with a volume of cell-derived content associatedwith the set of functionalized particles S330′; performing a stranddenaturing and second strand synthesis operation with the volume ofcell-derived content S335′; performing a cDNA amplification operationwith the volume of cell-derived content S340′; performing an mRNAparticle purification operation with PCR product of the cDNAamplification operation S345′; and performing a run completion operationupon completion of the mRNA particle purification operation S360′.

In more detail, performing a run preparation operation, S305′ caninclude sub-steps associated with one or more of: preparing a cellsuspension; initializing and performing operational checks of systemsubsystems (e.g., associated with the deck, associated with the gantry,associated with the base, etc.); returning the gantry to a homeposition; removing one or more seals from the reagent cartridge and/orloading reagents onto the reagent cartridge; positioning a sampleprocessing cartridge unit; removing one or more seals from the toolcontainer positioned at the deck; dispensing the cell suspension into astorage container prior to use; verifying proper positioning and states(e.g., in relation to expiration dates) of disposables for the protocol,upon scanning tags of disposables with a camera (e.g., machine visioncamera); receiving sample identification information (e.g., from anoperator); and initiating run of the sample. Steps of S305″ can beimplemented by the system automatically and/or by an operator.Furthermore, various sub-steps can be performed once, or repeated asrecommended.

In more detail, priming a sample processing cartridge of the system uponcompletion of the run preparation operation S310′ and co-capturing a setof cells of a sample with a set of functionalized particles, in singlecell format, at the sample processing cartridge S315′ can include one ormore of: dispensing a priming solution (e.g., in a manner that preventsbubbles from being trapped within the sample processing cartridge) intothe inlet reservoir of a sample processing cartridge; incubating thepriming solution within the sample processing cartridge; dispensing oneor more wash solutions into the inlet reservoir of the sample processingcartridge; transmitting solutions to a waste containment region of thesample processing cartridge; dispensing a cell suspension into the inletreservoir of the sample processing cartridge and capturing cells, insingle-cell format, within wells of the sample processing cartridge;dispensing a set of functionalized particles into the inlet reservoir ofthe sample processing cartridge and co-capturing the set offunctionalized particles with the set of cells; incubating content ofthe wells of the sample processing cartridge; and picking up/releasingvarious tools (e.g., by a gantry coupled to a pipette interface)involved with the substep(s). Steps S310′ and S315′ are preferablyperformed automatically by the system but can alternatively be performedin another suitable manner. Furthermore, various sub-steps can beperformed once, or repeated as recommended.

In more detail, performing a reverse transcription operation, at thesample processing cartridge, with lysate of the set of cells S320′ inthe presence of functionalized particles (e.g., barcoded microspheres)can include one or more of: dispensing one or more wash solutions intothe inlet reservoir of the sample processing cartridge; transmittingsolutions to a waste containment region of the sample processingcartridge; dispensing a particle-binding buffer into the inlet reservoirof the sample processing cartridge; dispensing a DTT solution into theinlet reservoir of the sample processing cartridge; dispensing a lysissolution into the inlet reservoir of the sample processing cartridge;displacing fluid above wells of the sample processing cartridge with anoil, thereby isolating contents of wells and preventing undesiredmaterial transfer across wells (e.g., as in U.S. application Ser. No.16/564,375 filed 9 Sep. 2019, which is herein incorporated in itsentirety by this reference); displacing the oil with air from the inletreservoir; dispensing a particle-binding wash buffer into the inletreservoir of the sample processing cartridge; dispensing a pre-RTreaction wash buffer into the inlet reservoir of the sample processingcartridge; dispensing a cDNA synthesis solution (e.g., SuperScript IV™)into the inlet reservoir of the sample processing cartridge; dispensingan RT cocktail into the inlet reservoir of the sample processingcartridge; incubating contents of the sample processing cartridge;performing incubation steps; and picking up/releasing various toolsinvolved with the substep(s). Step S320′ is preferably performedautomatically by the system but can alternatively be performed inanother suitable manner. Furthermore, various sub-steps can be performedonce, or repeated as recommended.

In more detail, retrieving the set of functionalized particles, withassociated bound target content, from the sample processing cartridgeS325′ can include performing magnetic separation operations (e.g., asdescribed above), using manual or automatic operations. Furthermore,various sub-steps can be performed once, or repeated as recommended.

In more detail, performing an exonuclease treatment operation with avolume of cell-derived content associated with the set of functionalizedparticles S330′ can include one or more of: mixing water and exonucleasebuffer to produce an exonuclease solution having a desiredconcentration; dispensing the exonuclease solution, with functionalizedparticles into a first PCR container; dispensing an oil (e.g., mineraloil) into the first PCR container; thermocycling and incubating contentsof the first PCR container; extracting a product of the first PCRcontainer; performing a separation operation with the product of thefirst PCR container; discarding waste from the separation operation;performing incubation steps; and picking up/releasing various toolsinvolved with the substep(s). Step S330′ is preferably performedautomatically by the system but can alternatively be performed inanother suitable manner. Furthermore, various sub-steps can be performedonce, or repeated as recommended.

In more detail, performing a strand denaturing and second strandsynthesis operation S335′ can include one or more of: transferring ahydroxide solution (e.g., sodium hydroxide solution) to a secondmagnetic separation container; mixing contents of the second magneticseparation container; activating a magnetic separation subsystem (e.g.,set of magnets coupled to actuator, described above) in proximity to thesecond magnetic separation container, thereby separating functionalizedmagnetic particles toward the magnet(s); discarding waste material fromthe second magnetic separation container; dispensing a wash solution tothe second magnetic separation container; mixing a second strandsynthesis primer enzyme within a process container in proximity to thesecond magnetic separation container; mixing the second strand synthesisprimer enzyme with contents of the second magnetic separation container;dispensing product of the second magnetic separation container into asecond PCR container; thermocycling contents of the second PCRcontainer, with mixing; transferring product of the second PCR containerto a third magnetic separation container; magnetically separatingproduct of the third magnetic separation container from waste anddiscarding waste; transferring a wash solution to the third magneticseparation container; and picking up/releasing various tools involvedwith the substep(s). Step S335′ is preferably performed automatically bythe system but can alternatively be performed in another suitablemanner. Furthermore, various sub-steps can be performed once, orrepeated as recommended.

In more detail, performing a cDNA synthesis operation S340′ can includeone or more of: mixing a polymerase blend (e.g., Kapa Biosystems HiFiHotstart Ready Mix™) within a cold storage volume; mixing the PCR mastermix from the cold storage volume with contents of the third magneticseparation container; aliquoting contents of the third magneticseparation container into a third, fourth, fifth, and sixth PCRoperation containers; aliquoting an oil (e.g., mineral oil) into thethird, fourth, fifth, and sixth PCR operation containers; running athird PCR operation; and picking up/releasing various tools involvedwith the substep(s). Step S340′ is preferably performed automatically bythe system but can alternatively be performed in another suitablemanner. Furthermore, various sub-steps can be performed once, orrepeated as recommended.

In more detail, performing an mRNA particle purification operation withPCR product of the cDNA amplification operation S345′ can include one ormore of: dispensing product from the third, fourth, fifth, and sixth PCRoperation containers into a fourth magnetic separation container; mixingand transferring PCR purification particles (e.g., AMPure beads XP™)into the fourth magnetic separation container; diluting and mixingethanol with nuclease-free water within a tenth magnetic separationcontainer; removing waste from the tenth magnetic separation container,after incubation; mixing nuclease-free water with target content of thetenth magnetic separation container; magnetically separating targetmRNA-cDNA product from tenth magnetic separation container; transferringtarget mRNA-cDNA product from tenth magnetic separation container tocold storage; and picking up/releasing various tools involved with thesubstep(s). Step S345 ′ is preferably performed automatically by thesystem but can alternatively be performed in another suitable manner.Furthermore, various sub-steps can be performed once, or repeated asrecommended.

In more detail, performing a run completion operation upon completion ofthe mRNA particle purification operation S360′ can include one or moreof: returning the gantry to a home configuration; providing anotification that sample processing is complete; releasing the reagentcartridge and/or sample processing cartridge from the system foroff-boardstorage; discarding system waste; performing a system cleaningoperation; and transitioning the system to a deactivated (e.g., idle,off) state. Steps of S360′ can be implemented by the systemautomatically and/or by an operator. Furthermore, various sub-steps canbe performed once, or repeated as recommended.

Example details of steps of the method 300′ are further described inTABLE 1 of Appendix A. In relation to steps of the method 300′,descriptions of ambient temperature and chilled reagents, as well aspositions associated with storage volumes of an embodiment of thereagent cartridge described above are described in TABLE 2 of AppendixA. In relation to TABLE 2 of Appendix A, volumes of reagents can bescaled according to sizes of sample processing chips used and/or numberof reactions run. In relation to magnetic separation operations,descriptions of apparatus and associated reagents used, as well aspositions associated with storage volumes of an embodiment of thereagent cartridge described above are described in TABLE 3 of AppendixA. In relation to amplification (e.g., PCR) operations, descriptions ofapparatus and associated reagents used, as well as positions associatedwith storage volumes of an embodiment of the reagent cartridge describedabove are described in TABLE 4 of Appendix A. In relation to fluidaspiration and delivery operations, descriptions of apparatus, as wellas positions associated with an embodiment of the tool containerdescribed above are described in TABLE 5 of Appendix A. In relation toactuation of components for automation of protocol aspects, gantry armand pipettor operations (e.g., in relation to apparatus coupling withdisposables, apparatus uncoupling from disposables, fluid mixing, wastediscarding, aspiration, delivery, aliquoting, etc.) are described inTABLE 6 of Appendix A. In relation to transitioning between modes forautomation of protocol aspects, sample processing cartridge operationsare described in TABLE 7 of Appendix A. In relation to transitioningbetween modes for automation of protocol aspects, heating and coolingsubsystem operation modes are described in TABLE 8 of Appendix A. Inrelation to transitioning between modes for automation of protocolaspects, magnetic separation subsystem operations are described in TABLE9 of Appendix A. In relation to amplification operations, PCR programdetails associated with the method 300′ are described in TABLE 10 ofAppendix A.

3.2 Method—Example Workflow for Single Cell Cytometry

As shown in FIG. 17 , a variation of the method, configured for singlecell cytometry 300″ can include: performing a run preparation operation,wherein the run preparation operation configures a system for performinga single cell cytometry protocol S305″; priming a sample processingcartridge of the system upon completion of the run preparation operationS310″; co-capturing a set of cells of a sample with a set offunctionalized particles, in single cell format, at the sampleprocessing cartridge S315″; lysing the set of cells followed by bindingreleased antibody-tagged oligonucleotides from the lysed cells tofunctionalized particles (e.g., barcoded microspheres) S320″(e.g., withwashing the unbound materials and then performing a reversetranscription operation); retrieving the set of functionalizedparticles, with associated bound target content, from the sampleprocessing cartridge S325″; performing an exonuclease treatmentoperation with a volume of cell-derived content associated with the setof functionalized particles S330″; performing a strand denaturing andsecond strand synthesis operation with the volume of cell-derivedcontent S335″; performing a cDNA amplification operation with the volumeof cell-derived content S340″; performing a particle purificationoperation with PCR product of the cDNA amplification operation S345″;performing a set of antibody-derived tag purification and amplificationoperations with outputs of the particle purification operation S350″;performing a set of mRNA purification and amplification operations withoutputs of the set of antibody-derived tag purification andamplification operations S355″; and performing a run completionoperation upon completion of the set of mRNA purification andamplification operations S360″.

In more detail, performing a run preparation operation, S305″ caninclude sub-steps associated with one or more of: preparing a cellsuspension; initializing and performing operational checks of systemsubsystems (e.g., associated with the deck, associated with the gantry,associated with the base, etc.); returning the gantry to a homeposition; removing one or more seals from the reagent cartridge and/orloading reagents onto the reagent cartridge; positioning a sampleprocessing cartridge unit; removing one or more seals from the toolcontainer positioned at the deck; dispensing the cell suspension into astorage container prior to use; verifying proper positioning and states(e.g., in relation to expiration dates) of disposables for the protocol,upon scanning tags of disposables with a camera (e.g., machine visioncamera); receiving sample identification information (e.g., from anoperator); and initiating run of the sample. Steps of S305 ″ can beimplemented by the system automatically and/or by an operator.Furthermore, various sub-steps can be performed once, or repeated asrecommended.

In more detail, priming a sample processing cartridge of the system uponcompletion of the run preparation operation S310″ and co-capturing a setof cells of a sample with a set of functionalized particles, in singlecell format, at the sample processing cartridge S315″ can include one ormore of: dispensing a priming solution into the inlet reservoir of asample processing cartridge; incubating the priming solution within thesample processing cartridge; dispensing one or more wash solutions intothe inlet reservoir of the sample processing cartridge; transmittingsolutions to a waste containment region of the sample processingcartridge; dispensing a cell suspension into the inlet reservoir of thesample processing cartridge and capturing cells, in single-cell format,within wells of the sample processing cartridge; dispensing a set offunctionalized particles into the inlet reservoir of the sampleprocessing cartridge and co-capturing the set of functionalizedparticles with the set of cells; incubating content of the wells of thesample processing cartridge; and picking up/releasing various toolsinvolved with the substep(s). Steps S310″ and S315″ are preferablyperformed automatically by the system but can alternatively be performedin another suitable manner. Furthermore, various sub-steps can beperformed once, or repeated as recommended.

In more detail, performing a reverse transcription operation, at thesample processing cartridge, with lysis of the set of cells S320″ caninclude one or more of: dispensing one or more wash solutions into theinlet reservoir of the sample processing cartridge; transmittingsolutions to a waste containment region of the sample processingcartridge; dispensing a particle-binding buffer into the inlet reservoirof the sample processing cartridge; dispensing a DTT solution into theinlet reservoir of the sample processing cartridge; dispensing a lysissolution into the inlet reservoir of the sample processing cartridge;displacing fluid above wells of the sample processing cartridge with anoil, thereby isolating contents of wells and preventing undesiredmaterial transfer across wells (e.g., as in U.S. application Ser. No.16/564,375 filed 9 Sep. 2019, which is herein incorporated in itsentirety by this reference); displacing the oil with air from the inletreservoir; dispensing a particle-binding wash buffer into the inletreservoir of the sample processing cartridge; dispensing a pre-RTreaction wash buffer into the inlet reservoir of the sample processingcartridge; dispensing a cDNA synthesis solution (e.g., SuperScript IV™)into the inlet reservoir of the sample processing cartridge; dispensingan RT cocktail into the inlet reservoir of the sample processingcartridge; incubating contents of the sample processing cartridge;performing incubation steps; and picking up/releasing various toolsinvolved with the substep(s). Step S320″ is preferably performedautomatically by the system but can alternatively be performed inanother suitable manner. Furthermore, various sub-steps can be performedonce, or repeated as recommended.

In more detail, retrieving the set of functionalized particles, withassociated bound target content, from the sample processing cartridgeS325″ can include performing magnetic separation operations (e.g., asdescribed above), using manual or automatic operations. Furthermore,various sub-steps can be performed once, or repeated as recommended.

In more detail, performing an exonuclease treatment operation with avolume of cell-derived content associated with the set of functionalizedparticles S330″ can include one or more of: mixing water and exonucleasebuffer to produce an exonuclease solution having a desiredconcentration; dispensing the exonuclease solution, with functionalizedparticles into a first PCR container; dispensing an oil (e.g., mineraloil) into the first PCR container; thermocycling and incubating contentsof the first PCR container; extracting a product of the first PCRcontainer; performing a separation operation with the product of thefirst PCR container; discarding waste from the separation operation;performing incubation steps; and picking up/releasing various toolsinvolved with the substep(s). Step S330″ is preferably performedautomatically by the system but can alternatively be performed inanother suitable manner. Furthermore, various sub-steps can be performedonce, or repeated as recommended.

In more detail, performing a strand denaturing and second strandsynthesis operation S335″ can include one or more of: transferring ahydroxide solution (e.g., sodium hydroxide solution) to a secondmagnetic separation container; mixing contents of the second magneticseparation container; activating a magnetic separation subsystem (e.g.,set of magnets coupled to actuator, described above) in proximity to thesecond magnetic separation container, thereby separating functionalizedmagnetic particles toward the magnet(s); discarding waste material fromthe second magnetic separation container; dispensing a wash solution tothe magnetic separation container; mixing a second strand synthesisprimer enzyme within a process container in proximity to the secondmagnetic separation container; mixing the second strand synthesis primerenzyme with contents of the second magnetic separation container;dispensing product of the second magnetic separation container into asecond PCR container; thermocycling contents of the second PCRcontainer, with mixing; transferring product of the second PCR containerto a third magnetic separation container; magnetically separatingproduct of the third magnetic separation container from waste anddiscarding waste; transferring a wash solution to the third magneticseparation container; and picking up/releasing various tools involvedwith the substep(s). Step S335″ is preferably performed automatically bythe system but can alternatively be performed in another suitablemanner. Furthermore, various sub-steps can be performed once, orrepeated as recommended.

In more detail, performing a cDNA synthesis operation S340″ can includeone or more of: mixing a polymerase blend (e.g., Kapa Biosystems HiFiHotstart Ready Mix™) within a cold storage volume; mixing the PCR mastermix from the cold storage volume with contents of the third magneticseparation container; aliquoting contents of the third magneticseparation container into a third, fourth, fifth, and sixth PCRoperation containers; aliquoting an oil (e.g., mineral oil) into thethird, fourth, fifth, and sixth PCR operation containers; running afirst PCR operation; and picking up/releasing various tools involvedwith the substep(s). Step S340″ is preferably performed automatically bythe system but can alternatively be performed in another suitablemanner. Furthermore, various sub-steps can be performed once, orrepeated as recommended.

In more detail, performing a particle purification operation with PCRproduct of the cDNA amplification operation S345″ can include one ormore of: dispensing product from the third, fourth, fifth, and sixth PCRoperation containers into a fourth magnetic separation container;separating target content of the fourth magnetic separation containerand transmitting target content into a fifth magnetic separationcontainer; mixing and transferring PCR purification particles (e.g.,AMPure beads XP™) into the fifth magnetic separation container;transferring product of the fifth magnetic separation container into asixth separation container; and picking up/releasing various toolsinvolved with the substep(s). Step S345″ is preferably performedautomatically by the system but can alternatively be performed inanother suitable manner. Furthermore, various sub-steps can be performedonce, or repeated as recommended.

In more detail, performing a set of antibody-derived tag purificationand amplification operations with outputs of the particle purificationoperation S350″ can include one or more of: mixing and transferring PCRpurification particles (e.g., AMPure beads XP™) into the sixth magneticseparation container; discarding waste from the sixth magneticseparation container; diluting and mixing ethanol with nuclease-freewater within a process container; transferring ethanol to the sixthmagnetic separation container; discarding waste from the sixth magneticseparation container, upon performing magnetic separation; transferringtarget content of the sixth magnetic separation container into a seventhmagnetic separation container; mixing PCR purification particles (e.g.,AMPure beads XP™) into the seventh magnetic separation container;transferring ethanol to the seventh magnetic separation container;discarding waste from the seventh magnetic separation container, uponperforming magnetic separation; dispensing water into the seventhmagnetic separation container; transferring purified cDNA from theseventh magnetic separation container into a seventh PCR operationcontainer; and picking up/releasing various tools involved with thesubstep(s). Step S350″ is preferably performed automatically by thesystem but can alternatively be performed in another suitable manner.Furthermore, various sub-steps can be performed once, or repeated asrecommended.

In more detail, performing a set of mRNA purification and amplificationoperations with outputs of the set of antibody-derived tag purificationand amplification operations S355″ can include one or more of:transferring PCR indexing primers into the seventh PCR operationcontainer; transferring a polymerase blend into the seventh PCRoperation container; mixing contents of the seventh PCR operationcontainer; transferring an oil (e.g., mineral oil) into the seventh PCRoperation container; initiating a second PCR operation; transferring PCRproduct from the seventh PCR operation container to an eighth magneticseparation container; transferring PCR purification particles (e.g.,AMPure beads XP) into the eighth magnetic separation container;transferring ethanol to the eighth magnetic separation container;discarding waste from the eighth magnetic separation container, uponperforming magnetic separation; dispensing water into the eighthmagnetic separation container; transferring purified cDNA from theeighth magnetic separation container into a storage container; andpicking up/releasing various tools involved with the substep(s). StepS355″ is preferably performed automatically by the system but canalternatively be performed in another suitable manner. Furthermore,various sub-steps can be performed once, or repeated as recommended.

In more detail, performing a run completion operation upon completion ofthe set of mRNA purification and amplification operations S360″ caninclude one or more of: returning the gantry to a home configuration;providing a notification that sample processing is complete; releasingthe reagent cartridge and/or sample processing cartridge from the systemfor off-boardstorage; discarding system waste; performing a systemcleaning operation; and transitioning the system to a deactivated (e.g.,idle, off) state. Steps of S360″ can be implemented by the systemautomatically and/or by an operator. Furthermore, various sub-steps canbe performed once, or repeated as recommended.

Example details of steps of the method 300″ are further described inTABLE 1 of Appendix B. In relation to steps of the method 300″,descriptions of ambient temperature and chilled reagents, as well aspositions associated with storage volumes of an embodiment of thereagent cartridge described above are described in TABLE 2 of AppendixB. In relation to TABLE 2 of Appendix B, volumes of reagents can bescaled according to sizes of sample processing chips used and/or numberof reactions run. In relation to magnetic separation operations,descriptions of apparatus and associated reagents used, as well aspositions associated with storage volumes of an embodiment of thereagent cartridge described above are described in TABLE 3 of AppendixB. In relation to amplification (e.g., PCR) operations, descriptions ofapparatus and associated reagents used, as well as positions associatedwith storage volumes of an embodiment of the reagent cartridge describedabove are described in TABLE 4 of Appendix B. In relation to fluidaspiration and delivery operations, descriptions of apparatus, as wellas positions associated with an embodiment of the tool containerdescribed above are described in TABLE 5 of Appendix B. In relation toactuation of components for automation of protocol aspects, gantry armand pipettor operations (e.g., in relation to apparatus coupling withdisposables, apparatus uncoupling from disposables, fluid mixing, wastediscarding, aspiration, delivery, aliquoting, etc.) are described inTABLE 6 of Appendix B. In relation to transitioning between modes forautomation of protocol aspects, sample processing cartridge operationsare described in TABLE 7 of Appendix B. In relation to transitioningbetween modes for automation of protocol aspects, heating and coolingsubsystem operation modes are described in TABLE 8 of Appendix B. Inrelation to transitioning between modes for automation of protocolaspects, magnetic separation subsystem operations are described in TABLE9 of Appendix B. In relation to amplification operations, PCR programdetails associated with the method 300″ are described in TABLE 10 ofAppendix B.

3.3 Method—Example Workflow for CITE-Seq Protocol (Single CellMultiomics)

As shown in FIG. 18 , a variation of the method, configured for singlecell multiomics 300′″ can include: performing a run preparationoperation, wherein the run preparation operation configures a system forperforming a single cell multiomics protocol S305′″; priming a sampleprocessing cartridge of the system upon completion of the runpreparation operation S310′″; co-capturing a set of cells of a samplewith a set of functionalized particles, in single cell format, at thesample processing cartridge S315′″; at the sample processing cartridge,with lysis of the set of cells, binding mRNA and antibody tagged oligosfrom the lysed cells to the set of functionalized particles, withwashing of unbound materials followed by performing a reversetranscription reaction S320′″; retrieving the set of functionalizedparticles, with associated bound target content, from the sampleprocessing cartridge S325′″; performing an exonuclease treatmentoperation with a volume of cell-derived content associated with the setof functionalized particles S330′″; performing a strand denaturing andsecond strand synthesis operation with the volume of cell-derivedcontent S335′″; performing a cDNA amplification operation with thevolume of cell-derived content S340′″; performing a particlepurification operation with PCR product of the cDNA amplificationoperation S345′; performing a set of antibody-derived tag purificationand amplification operations with outputs of the particle purificationoperation S350′″; performing a set of purification and amplificationoperations with outputs of the set of antibody-derived tag purificationand amplification operations S355′″; performing a set of mRNA particlepurification and amplification operations S360′″; and performing a runcompletion operation upon completion of the set of mRNA purification andamplification operations S35′″.

In more detail, performing a run preparation operation, S305′″ caninclude sub-steps associated with one or more of: preparing a cellsuspension; initializing and performing operational checks of systemsubsystems (e.g., associated with the deck, associated with the gantry,associated with the base, etc.); returning the gantry to a homeposition; removing one or more seals from the reagent cartridge and/orloading reagents onto the reagent cartridge; positioning a sampleprocessing cartridge unit; removing one or more seals from the toolcontainer positioned at the deck; dispensing the cell suspension into astorage container prior to use; verifying proper positioning and states(e.g., in relation to expiration dates) of disposables for the protocol,upon scanning tags of disposables with a camera (e.g., machine visioncamera); receiving sample identification information (e.g., from anoperator); and initiating run of the sample. Steps of S305′″ can beimplemented by the system automatically and/or by an operator.Furthermore, various sub-steps can be performed once, or repeated asrecommended.

In more detail, priming a sample processing cartridge of the system uponcompletion of the run preparation operation S310′″ and co-capturing aset of cells of a sample with a set of functionalized particles, insingle cell format, at the sample processing cartridge S315′″ caninclude one or more of: dispensing a priming solution into the inletreservoir of a sample processing cartridge; incubating the primingsolution within the sample processing cartridge; dispensing one or morewash solutions into the inlet reservoir of the sample processingcartridge; transmitting solutions to a waste containment region of thesample processing cartridge; dispensing a cell suspension into the inletreservoir of the sample processing cartridge and capturing cells, insingle-cell format, within wells of the sample processing cartridge;dispensing a set of functionalized particles into the inlet reservoir ofthe sample processing cartridge and co-capturing the set offunctionalized particles with the set of cells; incubating content ofthe wells of the sample processing cartridge; and picking up/releasingvarious tools involved with the substep(s). Steps S310′″ and S315′″ arepreferably performed automatically by the system but can alternativelybe performed in another suitable manner. Furthermore, various sub-stepscan be performed once, or repeated as recommended.

In more detail, performing a reverse transcription operation, at thesample processing cartridge, with lysis of the set of cells S320′″ caninclude one or more of: dispensing one or more wash solutions into theinlet reservoir of the sample processing cartridge; transmittingsolutions to a waste containment region of the sample processingcartridge; dispensing a particle-binding buffer into the inlet reservoirof the sample processing cartridge; dispensing a DTT solution into theinlet reservoir of the sample processing cartridge; dispensing a lysissolution into the inlet reservoir of the sample processing cartridge;displacing fluid above wells of the sample processing cartridge with anoil, thereby isolating contents of wells and preventing undesiredmaterial transfer across wells (e.g., as in U.S application Ser. No.16/564,375 filed 9 Sep. 2019, which is herein incorporated in itsentirety by this reference); displacing the oil with air from the inletreservoir; dispensing a particle-binding wash buffer into the inletreservoir of the sample processing cartridge; dispensing a pre-RTreaction wash buffer into the inlet reservoir of the sample processingcartridge; dispensing a cDNA synthesis solution (e.g., SuperScript IV™)into the inlet reservoir of the sample processing cartridge; dispensingan RT cocktail into the inlet reservoir of the sample processingcartridge; incubating contents of the sample processing cartridge;performing incubation steps; and picking up/releasing various toolsinvolved with the substep(s). Step S320′″ is preferably performedautomatically by the system but can alternatively be performed inanother suitable manner. Furthermore, various sub-steps can be performedonce, or repeated as recommended.

In more detail, retrieving the set of functionalized particles, withassociated bound target content, from the sample processing cartridgeS325′″ can include performing magnetic separation operations (e.g., asdescribed above), using manual or automatic operations. Furthermore,various sub-steps can be performed once, or repeated as recommended.

In more detail, performing an exonuclease treatment operation with avolume of cell-derived content associated with the set of functionalizedparticles S330′″ can include one or more of: mixing water andexonuclease buffer to produce an exonuclease solution having a desiredconcentration; dispensing the exonuclease solution, with functionalizedparticles into a first PCR container; dispensing an oil (e.g., mineraloil) into the first PCR container; thermocycling and incubating contentsof the first PCR container; extracting a product of the first PCRcontainer; performing a separation operation with the product of thefirst PCR container; discarding waste from the separation operation;performing incubation steps; and picking up/releasing various toolsinvolved with the substep(s). Step S330′″ is preferably performedautomatically by the system but can alternatively be performed inanother suitable manner. Furthermore, various sub-steps can be performedonce, or repeated as recommended.

In more detail, performing a strand denaturing and second strandsynthesis operation S335′″ can include one or more of: transferring ahydroxide solution (e.g., sodium hydroxide solution) to a secondmagnetic separation container; mixing contents of the second magneticseparation container; activating a magnetic separation subsystem (e.g.,set of magnets coupled to actuator, described above) in proximity to thesecond magnetic separation container, thereby separating functionalizedmagnetic particles toward the magnet(s); discarding waste material fromthe second magnetic separation container; dispensing a wash solution tothe magnetic separation container; mixing a second strand synthesisprimer enzyme within a process container in proximity to the secondmagnetic separation container; mixing the second strand synthesis primerenzyme with contents of the second magnetic separation container;dispensing product of the second magnetic separation container into asecond PCR container; thermocycling contents of the second PCRcontainer, with mixing; transferring product of the second PCR containerto a third magnetic separation container; magnetically separatingproduct of the third magnetic separation container from waste anddiscarding waste; transferring a wash solution to the third magneticseparation container; and picking up/releasing various tools involvedwith the substep(s). Step S335′″ is preferably performed automaticallyby the system but can alternatively be performed in another suitablemanner. Furthermore, various sub-steps can be performed once, orrepeated as recommended.

In more detail, performing a cDNA synthesis operation S340″ can includeone or more of: mixing a polymerase blend (e.g., Kapa Biosystems HiFiHotstart Ready Mix™) within a cold storage volume; mixing the PCR mastermix from the cold storage volume with contents of the third magneticseparation container; aliquoting contents of the third magneticseparation container into a third, fourth, fifth, and sixth PCRoperation containers; aliquoting an oil (e.g., mineral oil) into thethird, fourth, fifth, and sixth PCR operation containers; running afirst PCR operation; and picking up/releasing various tools involvedwith the substep(s). Step S340′″ is preferably performed automaticallyby the system but can alternatively be performed in another suitablemanner. Furthermore, various sub-steps can be performed once, orrepeated as recommended.

In more detail, performing a particle purification operation with PCRproduct of the cDNA amplification operation S345′″ can include one ormore of: dispensing product from the third, fourth, fifth, and sixth PCRoperation containers into a fourth magnetic separation container;separating target content of the fourth magnetic separation containerand transmitting target content into a fifth magnetic separationcontainer; mixing and transferring PCR purification particles (e.g.,AMPure beads XP™) into the fifth magnetic separation container;transferring product of the fifth magnetic separation container into asixth separation container; and picking up/releasing various toolsinvolved with the substep(s). Step S345′″ is preferably performedautomatically by the system but can alternatively be performed inanother suitable manner. Furthermore, various sub-steps can be performedonce, or repeated as recommended.

In more detail, performing a set of antibody-derived tag purificationand amplification operations with outputs of the particle purificationoperation S350″ can include one or more of: mixing and transferring PCRpurification particles (e.g., AMPure beads XP™) into the sixth magneticseparation container; discarding waste from the sixth magneticseparation container; diluting and mixing ethanol with nuclease-freewater within a process container; transferring ethanol to the sixthmagnetic separation container; discarding waste from the sixth magneticseparation container, upon performing magnetic separation; transferringtarget content of the sixth magnetic separation container into a seventhmagnetic separation container; mixing PCR purification particles (e.g.,AMPure beads XP™) into the seventh magnetic separation container;transferring ethanol to the seventh magnetic separation container;discarding waste from the seventh magnetic separation container, uponperforming magnetic separation; dispensing water into the seventhmagnetic separation container; transferring purified cDNA from theseventh magnetic separation container into a seventh PCR operationcontainer; and picking up/releasing various tools involved with thesubstep(s). Step S350′″ is preferably performed automatically by thesystem but can alternatively be performed in another suitable manner.Furthermore, various sub-steps can be performed once, or repeated asrecommended.

In more detail, performing a set of purification and amplificationoperations with outputs of the set of antibody-derived tag purificationand amplification operations S355″ can include one or more of:transferring PCR indexing primers into the seventh PCR operationcontainer; transferring a polymerase blend into the seventh PCRoperation container; mixing contents of the seventh PCR operationcontainer; transferring an oil (e.g., mineral oil) into the seventh PCRoperation container; initiating a second PCR operation; transferring PCRproduct from the seventh PCR operation container to an eighth magneticseparation container; transferring PCR purification particles (e.g.,AMPure beads XP) into the eighth magnetic separation container;transferring ethanol to the eighth magnetic separation container;discarding waste from the eighth magnetic separation container, uponperforming magnetic separation; dispensing water into the eighthmagnetic separation container; transferring purified cDNA from theeighth magnetic separation container into a storage container; andpicking up/releasing various tools involved with the substep(s). StepS355′″ is preferably performed automatically by the system but canalternatively be performed in another suitable manner. Furthermore,various sub-steps can be performed once, or repeated as recommended.

In more detail, performing a set of mRNA particle purification andamplification operations S360′″ can include one or more of: transferringnuclease-free water into the fifth magnetic separation container, withmixing and incubation; transferring product of the fifth magneticseparation container to a ninth magnetic separation container; mixingand transferring PCR purification particles (e.g., AMPure beads XP™)into the ninth magnetic separation container; transferring ethanol intothe ninth magnetic separation container; removing waste from the ninthmagnetic separation container, after incubation; mixing nuclease-freewater with target content of the ninth magnetic separation container;mixing and incubating contents of the ninth magnetic separationcontainer; transferring purified cDNA product from the ninth magneticseparation container to an eighth PCR operation container; transferringPCR master mix for mRNA amplification into the eighth PCR operationcontainer; mixing contents of the eighth PCR operation container;transferring polymerase blend (e.g., Kapa Biosystems HiFi Hotstart ReadyMix) into the eighth PCR operation container; mixing contents of theeighth PCR operation container; transferring an oil (e.g., mineral oil)into the eighth PCR operation container; performing a third PCRoperation within the eighth PCR operation container; transferringproduct of the third PCR operation from the eighth PCR operationcontainer to a tenth magnetic separation container; transferring PCRpurification particles into the tenth magnetic separation container;mixing contents of the tenth magnetic separation container; transferringethanol to the tenth magnetic separation container; discarding wastefrom the tenth magnetic separation container; transferring nuclease freewater into the tenth magnetic separation container; mixing contents ofthe tenth magnetic separation container, with incubation; magneticallyseparating target mRNA-cDNA product from tenth magnetic separationcontainer; transferring target mRNA-cDNA product from tenth magneticseparation container to cold storage; and picking up/releasing varioustools involved with the substep(s). Step S360′″ is preferably performedautomatically by the system but can alternatively be performed inanother suitable manner. Furthermore, various sub-steps can be performedonce, or repeated as recommended.

In more detail, performing a run completion operation upon completion ofthe set of mRNA purification and amplification operations S365′″ caninclude one or more of: returning the gantry to a home configuration;providing a notification that sample processing is complete; releasingthe reagent cartridge and/or sample processing cartridge from the systemfor off-boardstorage; discarding system waste; performing a systemcleaning operation; and transitioning the system to a deactivated (e.g.,idle, off) state. Steps of S365′″ can be implemented by the systemautomatically and/or by an operator. Furthermore, various sub-steps canbe performed once, or repeated as recommended.

Example details of steps of the method 300′″ are further described inTABLE 1 of Appendix C. In relation to steps of the method 300′″,descriptions of ambient temperature and chilled reagents, as well aspositions associated with storage volumes of an embodiment of thereagent cartridge described above are described in TABLE 2 of AppendixC. In relation to TABLE 2 of Appendix C, volumes of reagents can bescaled according to sizes of sample processing chips used and/or numberof reactions run. In relation to magnetic separation operations,descriptions of apparatus and associated reagents used, as well aspositions associated with storage volumes of an embodiment of thereagent cartridge described above are described in TABLE 3 of AppendixC. In relation to amplification (e.g., PCR) operations, descriptions ofapparatus and associated reagents used, as well as positions associatedwith storage volumes of an embodiment of the reagent cartridge describedabove are described in TABLE 4 of Appendix C. In relation to fluidaspiration and delivery operations, descriptions of apparatus, as wellas positions associated with an embodiment of the tool containerdescribed above are described in TABLE 5 of Appendix C. In relation toactuation of components for automation of protocol aspects, gantry armand pipettor operations (e.g., in relation to apparatus coupling withdisposables, apparatus uncoupling from disposables, fluid mixing, wastediscarding, aspiration, delivery, aliquoting, etc.) are described inTABLE 6 of Appendix C. In relation to transitioning between modes forautomation of protocol aspects, sample processing cartridge operationsare described in TABLE 7 of Appendix C. In relation to transitioningbetween modes for automation of protocol aspects, heating and coolingsubsystem operation modes are described in TABLE 8 of Appendix C. Inrelation to transitioning between modes for automation of protocolaspects, magnetic separation subsystem operations are described in TABLE9 of Appendix C. In relation to amplification operations, PCR programdetails associated with the method 300″ are described in TABLE 1 0 ofAppendix C.

3.4 Method—Example Workflow for Library Preparation

As shown in FIG. 19 , a variation of the method, configured for librarypreparation 300″″ can include: performing a run preparation operation,wherein the run preparation operation configures a system for performinga library preparation protocol S305″″; performing a library preparationoperation upon completion of the run preparation operation S310″″;performing a library purification operation with outputs of the librarypreparation operation S315″″; and performing a run completion operationupon completion of the library purification operation S360″″.

In more detail, performing a run preparation operation S305″″ caninclude substeps associated with one or more of: quantifyingconcentration of product (e.g., DNA concentration of product producedfrom a prior protocol; thawing out a reagent cartridge for librarypreparation, in a frozen storage state; processing storage volumes ofthe reagent cartridge (e.g., by vortexing, by centrifugation etc.);diluting a sequencing adaptor (e.g., NEBNext Illumina™ adaptor) solutionwith a dilution buffer; returning the sequencing adaptor solution andpreviously removed storages to the reagent cartridge; performingoperational checks of system subsystems (e.g., associated with the deck,associated with the gantry, associated with the base, etc.); returningthe gantry to a home position; positioning the reagent cartridge at adeck of the system; removing one or more seals from the reagentcartridge and/or loading reagents onto the reagent cartridge;positioning a sample processing cartridge unit at a deck of the system;removing one or more seals from the tool container positioned at thedeck; receiving an operator-loaded container (e.g., at a storage volumeof the reagent cartridge) for performing the library preparationoperation); initializing a heating and cooling subsystem (e.g., with aninitial temperature set point); verifying proper positioning and states(e.g., in relation to expiration dates) of disposables for the protocol,upon scanning tags of disposables with a camera (e.g., machine visioncamera); receiving sample identification information (e.g., from anoperator); and initiating run of the sample. Steps of S305″″ can beimplemented by the system automatically and/or by an operator.Furthermore, various sub-steps can be performed once, or repeated asrecommended.

In more detail, performing a library preparation operation uponcompletion of the run preparation operation S310″″ can include sub-stepsassociated with one or more of: transferring diluted cDNA from theoperator-loaded tube into a first cold storage container containingbuffer; transferring contents of the first cold storage tube to a secondcold storage container; incubating contents of the second cold storagecontainer; transferring a cDNA mixture from a second ambient storagecontainer into a first PCR operation container; fragmenting content ofthe first PCR operation container upon performing thermocyclingoperations at the first PCR operation container; transferring fragmentedDNA from the first PCR operation container to a fourth cold storagecontainer, with mixing; transferring contents of the fourth cold storagecontainer to a fifth cold storage container with mixing; transferringdiluted adaptor from a third cold storage container to the fifth coldstorage container with mixing and incubation; transferring contents ofthe fifth cold storage container to a second PCR operation containerwith incubation; transferring contents of the second PCR operationcontainer to a sixth cold storage container with mixing; transferringcontents of the sixth cold storage container to the second PCR operationcontainer with incubation; transferring contents of the second sixthcold storage container to a second magnetic separation container;transferring PCR purification particles (e.g., AMPure beads XP) to thesecond magnetic separation container with mixing; discarding waste fromthe second magnetic separation container; transferring ethanol to thesecond magnetic separation container; discarding waste from the secondmagnetic separation container; transferring TE buffer to the secondmagnetic separation container with incubation and magnetic separation;transferring purified cDNA from the second magnetic separation containerto a third PCR operation container; transferring indexing PCR master mixto the third PCR operation container with mixing; performing a fourthPCR operation; performing mixing steps; performing incubation steps; andpicking up/releasing various tools involved with the substep(s). StepS310″″ is preferably performed automatically by the system but canalternatively be performed in another suitable manner. Furthermore,various sub-steps can be performed once, or repeated as recommended.

In more detail, performing a library purification operation with outputsof the library preparation operation S315″″ can include sub-stepsassociated with one or more of: transferring product of the fourth PCRoperation from the third PCR operation container to a third magneticseparation container with mixing; transferring PCR purificationparticles (e.g., AMPure Beads XP) to the third magnetic separationcontainer with mixing, incubation, and magnetic separation; transferringethanol to the third magnetic separation container with incubation;removing waste from the third magnetic separation container;transferring nuclease-free water to the third magnetic separationcontainer with mixing, incubation, and magnetic separation; transferringcontents of the third magnetic separation container to a fourth magneticseparation container with mixing; transferring PCR purificationparticles (e.g., AMPure Beads XP) to the fourth magnetic separationcontainer with mixing, incubation, and magnetic separation; removingwaste from the fourth magnetic separation container; transferringethanol to the fourth magnetic separation container with incubation;discarding waste from the fourth magnetic separation container;repeating steps for further purification, with transfer of targetmaterial from the fourth magnetic separation container to a fifthmagnetic separation container, to a sixth magnetic separation container;transferring nuclease-free water to the sixth magnetic separationcontainer with incubation and magnetic separation; transferring purifiedcDNA for library construction to an eighth cold storage container;performing mixing steps; performing incubation steps; and pickingup/releasing various tools involved with the sub-step(s). Step S315″″ ispreferably performed automatically by the system but can alternativelybe performed in another suitable manner. Furthermore, various sub-stepscan be performed once, or repeated as recommended.

In more detail, performing a run completion operation upon completion ofthe library purification operation S360′″″ can include one or more of:returning the gantry to a home configuration; providing a notificationthat sample processing is complete; releasing the reagent cartridgeand/or sample processing cartridge from the system for off-boardstorage;discarding system waste; performing a system cleaning operation; andtransitioning the system to a deactivated (e.g., idle, off) state. Stepsof S360″″ can be implemented by the system automatically and/or by anoperator. Furthermore, various sub-steps can be performed once, orrepeated as recommended.

Example details of steps of the method 300″″ are further described inTABLE 1 of Appendix D. In relation to steps of the method 300″″,descriptions of ambient temperature and chilled reagents, as well aspositions associated with storage volumes of an embodiment of thereagent cartridge described above are described in TABLE 2 of AppendixD. In relation to TABLE 2 of Appendix D, volumes of reagents can bescaled according to sizes of sample processing chips used and/or numberof reactions run. In relation to magnetic separation operations,descriptions of apparatus and associated reagents used, as well aspositions associated with storage volumes of an embodiment of thereagent cartridge described above are described in TABLE 3 of AppendixD. In relation to amplification (e.g., PCR) operations, descriptions ofapparatus and associated reagents used, as well as positions associatedwith storage volumes of an embodiment of the reagent cartridge describedabove are described in TABLE 4 of Appendix D. In relation to fluidaspiration and delivery operations, descriptions of apparatus, as wellas positions associated with an embodiment of the tool containerdescribed above are described in TABLE 5 of Appendix D. In relation toactuation of components for automation of protocol aspects, gantry armand pipettor operations (e.g., in relation to apparatus coupling withdisposables, apparatus uncoupling from disposables, fluid mixing, wastediscarding, aspiration, delivery, aliquoting, etc.) are described inTABLE 6 of Appendix D. In relation to transitioning between modes forautomation of protocol aspects, sample processing cartridge operationsare described in TABLE 7 of Appendix D. In relation to transitioningbetween modes for automation of protocol aspects, heating and coolingsubsystem operation modes are described in TABLE 8 of Appendix D. Inrelation to transitioning between modes for automation of protocolaspects, magnetic separation subsystem operations are described in TABLE9 of Appendix D. In relation to amplification operations, PCR programdetails associated with the method 300″ are described in TABLE 1 0 ofAppendix D.

The system embodiment(s) can, however, be configured to implement otherworkflows including variations of those described, and/or otherworkflows.

4. Conclusion

The FIGS. illustrate the architecture, functionality and operation ofpossible implementations of systems, methods and computer programproducts according to preferred embodiments, example configurations, andvariations thereof. In this regard, each block in the flowchart or blockdiagrams may represent a module, segment, or portion of code, whichcomprises one or more executable instructions for implementing thespecified logical function(s). It should also be noted that, in somealternative implementations, the functions noted in the block can occurout of the order noted in the FIGS. For example, two blocks shown insuccession may, in fact, be executed substantially concurrently, or theblocks may sometimes be executed in the reverse order, depending uponthe functionality involved. It will also be noted that each block of theblock diagrams and/or flowchart illustration, and combinations of blocksin the block diagrams and/or flowchart illustration, can be implementedby special purpose hardware-based systems that perform the specifiedfunctions or acts, or combinations of special purpose hardware andcomputer instructions.

As a person skilled in the art will recognize from the previous detaileddescription and from the figures and claims, modifications and changescan be made to the preferred embodiments of the invention withoutdeparting from the scope of this invention defined in the followingclaims.

1. (canceled)
 2. A system for processing a sample, the systemcomprising: a reagent cartridge; a deck configured to support andposition: a sample processing cartridge, comprising a set of wells, at asecond region of the first side, a lid configured to cover an accessregion over the set of wells of the sample processing cartridge, the lidcomprising an open mode in which the access region is uncovered and aclosed mode in which the access region is covered; and a gantry coupledto the deck and comprising: a set of tracks defining paths of movementalong a set of axes, within a three dimensional volume bound by thefirst side of the deck, for a pipette interface; a heating and coolingsubsystem comprising a thermal body configured to thermally interfacewith the sample processing cartridge; and a tool configured totransition the lid between an open position in which the set of wells isuncovered, and a closed position in which the set of wells is covered,with retention of the sample processing cartridge at the heating andcooling subsystem.
 3. The system of claim 2, wherein the samplecomprises nucleic acids.
 4. The system of claim 2, wherein the samplecomprises a cell suspension.
 5. The system of claim 2, wherein thereagent cartridge comprises materials for performing polymerase chainreaction (PCR) operations with the sample.
 6. The system of claim 2,wherein the reagent cartridge comprises materials for performing librarypreparation operations with the sample.
 7. The system of claim 2,wherein the reagent cartridge comprises materials for performing reversetranscription and cDNA synthesis.
 8. The system of claim 2, wherein thelid comprises a gasket configured to seal the access region of thesample processing cartridge in the closed position.
 9. The system ofclaim 2, wherein the lid comprises a latch configured to couple with thesample processing cartridge.
 10. The system of claim 2, wherein furthercomprising a camera coupled with a pipettor coupled to the gantry, thecamera having a field of view including objects that the pipettor isaligned with.
 11. The system of claim 2, further comprising a separationsubsystem comprising a set of magnets coupled to a magnet actuatorconfigured to transition the set of magnets between a retracted stateand an extended state, wherein in the extended state, the set of magnetspasses into an opening of the deck.
 12. A method comprising: retaining asample processing cartridge at a deck; wherein the sample processingcartridge comprises: a set of wells, and a lid configured to cover anaccess region over the set of wells of the sample processing cartridge,the lid comprising an open position in which the access region isuncovered and a closed position in which the access region is covered;transferring a sample to the set of wells of the sample processingcartridge; performing a set of operations in coordination withtransferring a set of reagents from a reagent cartridge to the set ofwells of the sample processing cartridge, wherein the set of operationscomprises: priming the sample processing cartridge upon dispensing apriming solution into the sample processing cartridge; performing areverse transcription operation, at the set of microwells of the sampleprocessing cartridge, upon nucleic acids of the sample; performing astrand denaturing and second strand synthesis operation with uponperformance of the reverse transcription operation; transitioning thelid between the open position and the closed position, with retention ofthe sample processing cartridge at the deck; and performing a cDNAamplification operation upon performance of the strand denaturing andsecond strand synthesis operation.
 13. The method of claim 12, furthercomprising binding released mRNA content of the sample to barcodedfunctionalized particles within the set of wells.
 14. The method ofclaim 12, wherein transitioning the lid between the open position andthe closed position comprises applying a force to the lid, therebyisolating the access region of the sample processing cartridge.
 15. Themethod of claim 12, wherein the deck is coupled to a gantry comprising aset of tracks defining paths of movement along a set of axes, the methodfurther comprising: with a camera coupled with a pipettor coupled to thegantry, scanning a tag of the sample processing cartridge.
 16. Themethod of claim 15, further comprising guiding movement of components ofthe gantry, to within 100 micron precision, with feedback using a cameraimage generated by the camera.
 17. The method of claim 12, furthercomprising performing a set of antibody-derived tag purification andamplification operations with outputs of the cDNA amplificationoperation, for performance of a CITE-seq single cell multiomicsprotocol.
 18. The method of claim 12, further comprising performing alibrary preparation operation with material of the sample, wherein thereagent cartridge contains a set of reagents for the library preparationoperation, and wherein performing the library preparation operationcomprises fragmenting and amplifying nucleic acid content of the sample.19. The method of claim 18, wherein performing the library preparationoperation comprises determining a concentration of a DNA component ofthe sample; based upon the concentration performing a dilution operationupon the sample; fragmenting cDNA material of the sample; and amplifyingfragmented cDNA of the sample.
 20. The method of claim 18, furthercomprising performing a library purification operation with outputs ofthe library preparation operation upon combining material from thelibrary preparation operation with PCR purification particles, by way ofa pipettor coupled to an actuator of a gantry coupled to the deck. 21.The method of claim 12, further comprising performing a separationoperation upon the sample, wherein performing the separation operationcomprises actuating a magnet toward magnetic separation reservoir at thedeck.